From the Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstraße 30, D-95447 Bayreuth, Germany, the § Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Universitätsstraße 150, D-44801 Bochum, Germany, and the ¶ Université Joseph Fourier et CNRS, UMR 5575, BP53, CERMO, F-38041 Grenoble cedex 9, France
Received for publication, September 23, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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We have recently discovered a protochlorophyllide
(Pchlide)-based light-harvesting complex involved in chlorophyll
a biosynthesis. This complex consists of the two previously
identified NADPH:protochlorophyllide oxidoreductases (PORs), PORA and
PORB, their natural substrates (Pchlide b and Pchlide
a, respectively), plus NADPH. These are all held together
in a stoichiometry of five PORA-Pchlide b-NADPH complexes
and one PORB-Pchlide a-NADPH complex in the prolamellar body of etioplasts. The assembly of this novel light-harvesting POR-Pchlide complex (LHPP) requires both the proper interaction of the
PORA and PORB with their cognate substrates as well as the
oligomerization of the resulting POR-pigment-NADPH ternary complexes
into the native, lipid-containing structure of the etioplast. In this
study, we demonstrate that the conserved extra sequence that
distinguishes PORA and PORB from the structurally related short-chain
alcohol dehydrogenases, is dispensable for pigment binding but needed
for the assembly of LHPP. As shown by in vitro mutagenesis,
deleting this extra sequence gave rise to assembly-incompetent but
pigment-containing PORA and PORB polypeptides.
Photosynthesis supports our life on Earth. During photosynthesis,
plants collect sunlight and convert this into chemical energy to be
conserved in ATP and NADPH (1). A key role in the processes of light
absorption and energy transduction is played by the light-harvesting complexes, in particular two light-harvesting chlorophyll
a/b-protein complexes termed
LHCI1 and LHCII (2).
LHCII is a trimer (3) that operates as the basic antenna to provide the
reaction center of photosystem II with excitation energy (4). LHCII is
conserved in all green plants (5). Monomeric LHCII consists of 232 amino acids that form three A similar principle of energy transfer has been proposed for the
prolamellar body of etioplasts (11). Prolamellar bodies are
paracrystalline structures that are present in etiolated plants (12).
They contain the immediate Chl precursors protochlorophyllide (Pchlide)
a and Pchlide b, respectively, which are
differentially bound to two forms of the photoenzyme NADPH:Pchlide
oxidoreductase, termed PORA and PORB (13). Based on in vitro
reconstitution experiments, we proposed that five PORA-Pchlide
b-NADPH complexes and one PORB-Pchlide a-NADPH
complex may structurally and functionally interact in terms of a novel
"light-harvesting POR-Pchlide" complex termed LHPP (11). Recent
work resolved a Pchlide a/b-containing PORA-PORB
complex from the prolamellar body of etioplasts (13). We repeatedly
observed that Pchlide b present in the native and in
vitro-reconstituted complexes was not photoconvertible in the first place and that only Pchlide a was reduced to Chlide
a (13). Taking into account previously documented energy
transfer reactions, taking place from photoinactive Pchlide to
photoactive Pchlide in the prolamellar body of etioplasts (14-19), we
hypothesized that Pchlide b may be operative as a light
scavenger (11, 13).
In addition to the pigment substrates NADPH and Pchlide, native LHPP is
presumed to contain galacto- and sulfolipids (11, 13). In our recent
in vitro reconstitution experiments, the lipids shifted the
spectral properties of the complex from around 630 to 650 nm (11, 13).
Hereby an overlap was established to the action spectrum of
phytochrome-dependent regulation of hypocotyl elongation
observed in vivo (20, 21).
The complex molecular architecture of LHPP and particularly the fact
that it contains two closely related POR proteins, which must bind and
properly interact with their cognate substrates in order to be
operative during Chl a biosynthesis, put tremendous constraints on the assembly pathway of LHPP. Initial studies suggested that the substrate-dependent import of the cytosolic
precursor of the PORA into the plastids might be a mechanism to provide assembly-competent PORA-Pchlide (b)-NADPH complexes
(22-25). Also PORB seemed to interact with Pchlide, presumably Pchlide
a, but this interaction was likely to take place only after
translocation (24). Import per se was Pchlide-independent
(24, 25).
Where and when PORA-Pchlide b-NADPH and PORB-Pchlide
a-NADPH ternary complexes interact with each other during
the establishment of LHPP is unknown; nor has it been determined which
structural elements in the PORA and PORB polypeptides are essential for
their oligomerization. As a first step to investigate the assembly
pathway of LHPP, we genetically deleted the extra sequence that
distinguishes PORA and PORB from the structurally related short-chain
alcohol dehydrogenases (26-28). As shown by in organello
and in vitro studies, this deletion in either case had no
effect on the import of the cytosolic POR precursors into the plastids,
their binding to Pchlide, and the stability of the imported, mature
PORA and PORB proteins inside the organelles. However, either
truncation completely abolished the assembly of LHPP in
vitro.
Primers--
Primer sequences were as follows: primer 1, 5'-AACTGCAGATGGGCAAGAAGACGCTGCGGCAG-3'; primer 2, 5'-AACTGCAGGGTGGATCATAGTCCGACGAGCTT-3'; primer 3, 5'-AACTGCAGATGGGCAAGAAGACTGTCCGCACG-3'; primer 4, 5'-AACTGCAGTGATCATGCGAGCCCGACGAGCTT-3'; primer 5, 5'-TTTGCCCATGGTAATGGAGCCGAC-GATGACCAT-3'; primer 6, 5'-AAAACCATGGGCGACGAGAGCTTCGACGGCGCC-3'; primer 7, 5'-TTTGCCCATGGTAATGGAGCCGACGATGATGAG-3'; primer 8, 5'-AAAACCATGGGCGCGGAGTTCGACGGCGCCAAG-3'.
Production of Truncated In Vitro Transcription/Translation--
Cell-free protein
synthesis was performed in a hand-made or TNT wheat germ-coupled
in vitro transcription/translation system (Promega GmbH,
Mannheim, Germany), according to the manufacturer's instructions (see
also Ref. 34). Precursors to be used for studying protein import were
denatured with 8 M urea and diluted to a final 0.2 M urea concentration immediately before use.
Protein Import and Postimport Treatments--
Protein import was
studied as described previously (22-24). Briefly, chloroplasts were
isolated from 5-day-old, light-grown plants by differential
centrifugation and Percoll density gradient centrifugation and further
purified on Percoll cushions. Import assays contained 7.5 µl of
complete pre-mix consisting of 25 µl of doubly concentrated, ATP-free
import buffer (22), 2 µl of a 125 mM stock solution of
Mg-ATP, pH 7.0, 10 µl of the indicated radiolabeled, urea-denatured
precursors, 3 µl of double-distilled water, and 0.5 µl of either a
10 mM stock solution of 5-aminolevulinic acid (5-ALA)
prepared in 10 mM phosphate buffer, pH 8.0, or phosphate buffer alone. The import reaction was initiated by the addition of 2 µl of the purified plastids (1 × 107), and five
samples were run and analyzed in parallel for each of the different
precursors. One assay was immediately stopped by the addition of a
doubly concentrated SDS sample buffer (see below), while the remaining
four samples were incubated at 23 °C in complete darkness for 15 min. Two of the latter four samples contained phosphate-buffered
5-ALA, whereas the other two contained only phosphate buffer. In either
case, one each of the different samples was treated with thermolysin
after the incubation (35), whereas the other was left untreated.
Postimport incubations were performed with protease-treated plastids
recovered from the import mixtures by centrifugation on Percoll, at
23 °C for 30 min either in darkness or white light. After the
addition of an equal volume of doubly concentrated SDS-sample buffer
(36) and boiling the samples for 2 min, protein was analyzed electrophoretically and detected by autoradiography (see below).
Reconstitution of POR-Pigment
Complexes--
[35S]Methionine-labeled PORA, PORB,
Protease Treatment of POR-Pigment Complexes in
Vitro--
Protease treatment of reconstituted POR-pigment-NADPH
complexes was performed as described previously (24) but in incubation mixtures containing 7.5 µl of doubly concentrated assay buffer (22),
1 µl of a 25 mM stock solution of Mg-ATP, pH 7.0, 5 µl of a plastid protease mixture prepared from barley chloroplasts (39),
and 1.5 µl of double-distilled water. After a 15-min incubation in
the dark, the assays were terminated by the addition of 1 µl of a
mixture containing 10 µg ml Assembly Assay of LHPP--
Different amounts of gel-filtered
PORA-Pchlide (ZnPP) b-NADPH, NADPH:protochlorophyllide oxidoreductase (EC 1.3.33.1) belongs to
the family of short-chain alcohol dehydrogenases (26-28). All plant
POR proteins characterized thus far, including the PORA and PORB of
barley used in this study, are conserved in their length, primary amino
acid sequence, and active site residues (27, 40, 41). However, they
differ from each other and the related short-chain alcohol
dehydrogenases in possessing variable NH2-terminal
extensions, referred to as transit peptides, which are required for
their post-translational transport into the plastids.
Another striking difference is the occurrence of a short stretch of
hydrophobic amino acids in the central region of the polypeptide (Fig.
1A), which distinguishes POR
from members of the alcohol dehydrogenase family, comprising
3
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices embedded into the thylakoid
membrane of the chloroplast (6), the site of photosynthesis. Twelve
chlorophylls (Chls) (seven Chl a and five Chl b
molecules) and two carotenoids (luteins) are bound per LHCII molecule
(6). In the center of the complex, Chl b is in close contact
with Chl a for rapid energy transfer and with the
carotenoids that prevent the formation of toxic singlet oxygen (6).
Energy transfer from Chl b to the closely related Chl
a (both compounds differ only in a formyl group instead of a
methyl group at the 7-position in the chlorin ring of Chl a) (for a review, see Ref. 7) can take place because of the different energy contents and decay times of their excited states (8, 9). Energy
absorbed by Chl b is transferred to Chl a within less than 1 ps, where it remains for 1-3 ns, as shown by the Chl a fluorescence decay time in the purified,
detergent-solubilized complex (10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(p)PORA and
(p)PORB
Derivatives--
cDNA clones MatA1.1 and MatB1.1, encoding the
mature PORA and PORB polypeptides lacking their
NH2-terminal transit peptides for import into chloroplasts,
were generated by a polymerase chain reaction-based approach (29),
using primers 1 plus 2 and primers 3 plus 4, in combination with clones
A7 (30) and L2 (31), respectively, as templates. cDNA clones
Mat
A1.1 and Mat
B1.1, encoding truncated PORA (
PORA) and PORB
(
PORB) molecules, respectively, were produced identically but with
two additional primer combinations: primers 1 and 5 plus primers 2 and
6 (pMat
A1.1) and primers 3 and 7 plus primers 4 and 8 (pMat
B1.1),
respectively. For construction of cDNA clones encoding full-length
PORA and PORB precursors lacking the extra loop (i.e.
pPORA and
pPORB), clones Mat
A1.1 and Mat
B1.1 were treated
with BamHI (32) and annealed with PCR-derived
BamHI-cut fragments encoding transA and transB (the transit
peptides of the pPORA and pPORB, respectively), which had been
amplified as described (25). The identity of all of the different
clones was confirmed by DNA sequencing, using the gel system described in Ref. 33.
PORA, and
PORB molecules were synthesized by coupled in
vitro transcription/translation of the recombinant clones
described above and resolved by SDS-PAGE (36). After electrophoresis
and autoradiography, the radioactivity bands were cut out and counted
in a liquid scintillation counter. After correcting the incorporation
rates for the different methionine contents, equal amounts of the
different proteins were supplemented with 0.5 mM NADPH and
incubated with either chemically synthesized Pchlide a and
Pchlide b or their zinc counterparts, ZnPPa and ZnPPb, respectively (37, 38), each added to 10 µM final concentrations to the assays (22).
POR-pigment-NADPH complexes formed during a 15-min dark incubation were
depleted of non-protein-bound pigments by gel filtration on Sephadex
G15, as described (22), and were then either kept in darkness or
illuminated with white light for 15 min. Enzymatic product formation
occurring during this preillumination was measured fluorimetrically
using acetone-extracted pigments.
1 antipain and 1 µg
ml
1 pepstatin, which efficiently block the POR-degrading
stromal protease (39).
PORA-Pchlide (ZnPP)
b-NADPH, PORB-Pchlide (ZnPP) a-NADPH, and
PORB-Pchlide (ZnPP) a-NADPH complexes were mixed in the
combinations given herein and incubated in the dark for 15 min.
Then one aliquot of the reaction mixtures was immediately precipitated
with trichloroacetic acid (11). Another aliquot was subjected to gel
filtration on Sephadex G100 or Superose 6 (Amersham Biosciences).
Individual fractions were harvested, and aliquots were taken for
radioactivity measurements in a liquid scintillation counter. Pooled
fractions were then treated with trichloroacetic acid as described
above and processed with acetone, ethanol, and diethyl ether; and
protein was resolved on 10-20% polyacrylamide gradients containing
SDS (36). The gel in Fig. 1B shows a separation on a 5-15%
polyacrylamide gradient containing SDS. After electrophoresis,
35S-labeled PORA-PORB higher molecular weight complexes and
nonassembled 35S-PORA and 35S-PORB were
visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,20
-hydroxysteroid dehydrogenase and dihydropteridine reductase
(42, 43). Wilks and Timko (27) proposed that this so-called "extra
loop" may be involved in substrate binding, subunit-subunit
interaction, or membrane association. In addition, an involvement in
protein import and postimport stabilization of the imported, mature
proteins could not be excluded.
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Fig. 1.
Production of authentic and truncated PORA
and PORB polypeptides. A, schematic representation of
the constructed pPOR polypeptides lacking the indicated internal
sequences, as compared with their authentic counterparts.
Light gray and dark gray
columns highlight the presence of the
NH2-terminal transit peptides, which are required for
posttranslational transport of the POR precursors into the plastids.
B, in vitro synthesis of pPORA, pPORB,
pPORA,
and
pPORB polypeptides by coupled transcription/translation of
corresponding recombinant clones and detection of the radiolabeled
products by SDS-PAGE and autoradiography. C, as in
B but showing mature POR polypetides lacking their
respective transit peptides.
To test these different possibilities, truncated precursor PORA
(pPORA) and precursor PORB (
pPORB) molecules, which lacked amino
acids 217-252 and 225-260, respectively, representing the extra loop
(Fig. 1A), were generated by a polymerase chain
reaction-based approach (see "Materials and Methods").
Subsequently, the resulting recombinant clones were used for in
vitro transcription/translation in the presence of
[35S]methionine (Fig. 1B). As a control to the
truncated precursors, the authentic, in vitro synthesized
pPORA and pPORB were used (Fig. 1, A and B).
Mature PORA and PORB polypeptides containing or lacking the extra loop
were produced in parallel (Fig. 1C).
The various radiolabeled precursors were denatured with urea (see "Materials and Methods") and added to barley chloroplasts that had been isolated from light-grown plants. Then the mixtures were incubated for 15 min in the dark with either 5-ALA dissolved in phosphate buffer or phosphate buffer alone. After this incubation, half of the assays were treated with thermolysin, whereas the other halves were left untreated.
When import of the authentic and truncated pPORA was compared, no
difference could be seen for 5-ALA-incubated, Pchlide-containing chloroplasts (Fig. 2A). In
case of the truncated pPORB, a slight drop of import was detectable as
compared with the authentic precursor (Fig. 2A). Precursor
molecules that had not been imported were degraded during postimport
thermolysin treatment (Fig. 2A, + 5-ALA; compare Thl and + Thl).
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In the case of chloroplasts that lacked the 5-ALA-derived Pchlide, no
import of the pPORA and its pPORA derivative was detectable (Fig.
2A). Consistent with previous results (22-25), the
precursor in either case remained quantitatively unchanged after the
incubation. It was rapidly degraded during subsequent thermolysin
treatment (Fig. 2A,
5-ALA; compare
Thl and + Thl). By contrast, import of the pPORB
and its
pPORB derivative into Pchlide-free chloroplasts was very
possible and did not require exogenous 5-ALA (Fig. 2A, compare + 5-ALA and
5-ALA).
We next asked whether the extra loop distinguishing the PORA and PORB from the related alcohol dehydrogenases might affect the stability of the imported and processed proteins. Replicate plastid samples, which had been treated with thermolysin after import, were repurified on Percoll and subsequently incubated at 23 °C either in white light or darkness for an additional 30-min period.
Fig. 2B shows that postimport incubations in darkness had no effect on the stability of the imported and processed enzymes. In all cases, identical levels of the mature POR proteins were maintained. When the samples were illuminated, all imported POR proteins rapidly vanished, however (Fig. 2C). Most likely as a result of light-induced chlorophyllide formation (39), the different imported proteins were degraded by plastid proteases (Fig. 2, compare B and C).
We next determined the turnover rates of in
vitro-reconstituted POR-pigment complexes, using a stromal
protease isolated from barley chloroplasts (39). We assumed that this
type of assay might unveil minor differences in the stability of the
truncated and nontruncated POR proteins. Mature PORA and PORB
polypeptides lacking both their extra loops and chloroplast transit
peptides were produced as described in the legend to Fig. 1C
and purified by glycerol gradient centrifugation. Then equal amounts of
the two truncated and two authentic POR polypeptides were reconstituted into ()POR-pigment-NADPH complexes (22). PORA and
PORA were incubated for 15 min in the dark with Pchlide b and NADPH,
whereas PORB and
PORB were supplemented with Pchlide a
and NADPH, respectively. After a subsequent step of gel filtration on
Sephadex G15 to remove non-protein-bound pigments, the fluorescence
properties of the recovered (
)POR-Pchlide-NADPH ternary complexes
were determined in acetone (22).
When the levels of PORA- and PORA-bound Pchlide b were
compared, almost no difference was observed; PORA and
PORA bound practically indistinguishable amounts of the pigment (Fig.
3A, solid
versus dashed line, respectively).
Similar results were obtained for the PORB and
PORB, which bound
almost the same levels of Pchlide a (Fig. 3B,
solid and dashed line, respectively).
Exposing the different samples to white light in all cases caused
enzymatic chlorophyllide formation (data not shown, but see
accompanying paper (13)), demonstrating that the
PORA and
PORB
polypeptides were enzymatically active.
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As a next step, we tested the stability of the reconstituted
POR-Pchlide-NADPH and POR-chlorophyllide-NADP+ ternary
complexes. As a control to the pigment-complexed proteins, the naked
PORA, PORA,
PORB, and PORB polypeptides were used. Then a stromal
protease that had been prepared from barley chloroplasts (39) was
added. After various time intervals, aliquots were taken and
precipitated with trichloroacetic acid, and protein was analyzed by
SDS-PAGE.
Fig. 4 shows time courses of the amount
of the PORA and PORB and their PORA and
PORB derivatives. In the
presence of their cognate substrates, all four proteins were stabilized
to similar extents (Fig. 4, A-D, solid
lines). In the absence of Pchlide b and Pchlide
a, respectively, and NADPH, all four polypeptides were
rapidly degraded, however (Fig. 4, A-D, dashed
lines). Light-induced Chlide formation, allowed to proceed
during a preincubation (22, 23), promoted this decline (Fig. 4,
A-D, dotted lines, respectively).
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We next asked whether the extra loop might be involved in the
formation of higher molecular weight PORA-PORB supracomplexes. PORA-Pchlide b-NADPH, PORA-Pchlide b-NADPH,
PORB-Pchlide a-NADPH, and PORB-Pchlide a-NADPH
ternary complexes were produced from the authentic and truncated PORA
and PORB polypeptides, NADPH, Pchlide b, and Pchlide
a, respectively, and purified by gel filtration on Sephadex
G15 and subsequent centrifugation on glycerol gradients. The four
different ternary complexes were then mixed in the combinations given
in Fig. 5A and incubated for
an additional 15-min period in darkness. All of the 16 different assay
mixtures in turn were subjected to gel filtration (see "Materials and
Methods"). Individual fractions were harvested, and the radioactivity
of each aliquot was determined by liquid scintillation counting.
Fractions containing higher molecular weight
35S-(
)PORA-(
)PORB supracomplexes or nonassembled free
subunits were pooled and analyzed by SDS-PAGE and autoradiography
(13).
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Fig. 5B shows the autoradiograms of an experiment in which
equimolar combinations of the four different POR-pigment-NADPH complexes were mixed and allowed to form higher molecular weight complexes. It turned out that only POR ternary complexes containing the
nontruncated, authentic PORA plus PORB were positive (Fig. 5B, row b, fraction
5). In all of the other cases, no such supracomplexes were
formed. The free, nonassembled subunits were recovered in fractions 17 and 18 (Fig. 5B). Varying the initial concentration of the
()PORA-Pchlide b-NADPH and (
)PORB-Pchlide
a-NADPH had no impact on this negative result.
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DISCUSSION |
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POR was assigned to be a member of the short-chain alcohol dehydrogenase family (26-28). Its high overall similarity in secondary structure was recently used to construct homology models for POR of Synechocyctis (44) and pea (45). On the basis of these models, predictions were made of which amino acid residues might be essential for substrate and cofactor binding as well as membrane association (44, 45).
POR appears to consist of a central parallel -sheet that is composed
of seven
-strands (
-1 to
-7) surrounded by eight (pea) or nine
(Synechocystis)
-helices (
-A to
-I) (44, 45). Just
before the highly conserved YXXXK motif, which is located in
helix
-F forming one side of the active site, a 35-amino acid insertion (33 amino acids in case of Synecocystis POR) was
predicted (44, 45). This extra loop is not present in most of the other members of the short-chain alcohol dehydrogenase family (42, 43, 46),
but it is found in human carbonyl reductase as a 41-residue insertion
at an equivalent position (26).
In the present study, an in vitro mutagenesis approach was used to address the question of which role the extra loop present in the PORA and PORB of barley might play. We demonstrate that deleting this loop, which (with a few exceptions) is almost identical in amino acid sequence in the PORA and PORB (see Fig. 1), had no impact on protein import, the stability of the imported, processed enzymes, or the pigment binding properties of the mature proteins (Figs. 2-4). Rather, either truncation likewise abolished the in vitro assembly of higher molecular weight PORA/PORB-Pchlide complexes indicative of LHPP (Fig. 5).
The extra loop does not display homology to other sequence motifs found in the data banks (even not to the aforementioned analogous sequence of the human carbonyl reductase). Among the suggestions for what might be the putative function of this sequence is the modulation of intermolecular interactions (27, 44, 45). This was doubted recently, however, since human carbonyl reductase and POR can be active in their monomeric states (44). On the other hand, it was shown by cross-linking experiments with isolated prolamellar bodies of wheat that POR occurs in aggregates that are mainly dimers (47). Already in 1962, a high molecular mass complex of ~600 kDa was described to be part of the bean Pchlide holochrome (48) that may represent pigment-complexed POR aggregates. More recent studies with pea POR expressed as a fusion with maltose-binding protein showed that POR is able to form stable active dimers (49). We were able to reconstitute a multimeric complex (LHPP) consisting of five PORA-Pchlide b-NADPH ternary complexes and one PORB-Pchlide a-NADPH ternary complex that interacted with isolated galacto- and sulfolipids. This complex has apparently the same fluorescence spectroscopic properties as isolated prolamellar bodies (see accompanying paper (13)). It is therefore safe to assume that a similar complex may exist in vivo.
Our results clearly favor an involvement of the extra loop in protein-protein interactions. This would be true at least for the situation found in etioplasts where both PORA and PORB are present in barley (31) and Arabidopsis thaliana (50). As demonstrated in this article, we observed that deleting the extra sequence in either PORA or PORB gave rise to assembly-incompetent ternary complexes. Interestingly, we did not observe self-oligomerization between either PORA-Pchlide b-NADPH and PORB-Pchlide a-NADPH ternary complexes into higher molecular mass supracomplexes. This finding points to a highly specific, although indirect, function of the extra loop in mediating PORA-PORB-interactions.
Birve et al. (28) put forth the idea that the extra loop may
be involved in membrane binding. The authors proposed that the extra
loop may cooperate with other regions of the polypeptide, in particular
amphipathic segments containing tryptophan. Of the four tryptophan
residues present in POR, one is in the so-called Rossman fold, which
represents the binding site of NADPH, two are in residues 349-353 of a
-sheet structure, and the fourth is located in residues 371-385 of
an
-helical region (28). Each of these tryptophan residues could
contact the lipid polar head groups of respective target membranes. In
such a scenario, the amphipathic helices could then become orientated
parallel to the water-lipid interface of the membrane, allowing the
hydrophobic amino acids to face the hydrophobic core of the bilayer and
the hydrophilic residues to reside in the aqueous environment. If these
interactions were to occur in case of POR, its membrane binding would
resemble that of prostaglandin H synthase, which is anchored into the
lipid bilayer via four amphipathic helices containing tryptophan
(51-53).
The model proposed of Birve et al. (28) is attractive. Since it rests on the assumption that PORA and PORB interact each independently with the membrane, it would be appropriate for the integration of PORB into thylakoids of chloroplast. In the case of the association of both the PORA and PORB with the prolamellar bodies of etioplasts, however, it would be too simple. As explained before, PORA and PORB interact to form supramolecular complexes associated with the lipids of the prolamellar body.
Although the results presented here clearly indicate a role of the
extra loop in protein-protein interactions, much more work is needed to
obtain information on the precise function of this structure in
vivo.
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ACKNOWLEDGEMENT |
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Part of this work was performed in the Department of Prof. Dr. E. W. Weiler at the Institute for Plant Physiology, Ruhr-Universität Bochum, Bochum, Germany. We are grateful to Prof. Dr. Weiler for support of the work.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant RE1465/1-1,1-2 (to C. R.).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.
To whom correspondence should be addressed: Lehrstuhl für
Pflanzenphysiologie, Universität Bayreuth, Universitätsstr.
30, 95447 Bayreuth, Germany. Tel.: 49-921-55-26-27; Fax:
49-921-75-77-442; E-mail: christiane.reinbothe@uni-bayreuth.de.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M209739200
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ABBREVIATIONS |
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The abbreviations used are:
LHC, light-harvesting chlorophyll a/b-protein complex;
5-ALA, 5-aminolevulinic acid;
LHPP, light-harvesting POR-Pchlide
complex;
Pchlide, protochlorophyllide;
Chlide, chlorophyllide;
Chl, chlorophyll;
POR, NADPH:protochloro-phyllide oxidoreductase;
()POR, truncated POR;
ZnPP, zinc protopheophorbide.
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REFERENCES |
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