The Extra Loop Distinguishing POR from the Structurally Related Short-chain Alcohol Dehydrogenases Is Dispensable for Pigment Binding but Needed for the Assembly of Light-harvesting POR-Protochlorophyllide Complex*

Christiane ReinbotheDagger, Anja Lepinat, Markus Deckers§, Erwin Beck, and Steffen Reinbothe§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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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 alpha -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).

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.

    MATERIALS AND METHODS
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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 Delta (p)PORA and Delta (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 MatDelta A1.1 and MatDelta B1.1, encoding truncated PORA (Delta PORA) and PORB (Delta PORB) molecules, respectively, were produced identically but with two additional primer combinations: primers 1 and 5 plus primers 2 and 6 (pMatDelta A1.1) and primers 3 and 7 plus primers 4 and 8 (pMatDelta B1.1), respectively. For construction of cDNA clones encoding full-length PORA and PORB precursors lacking the extra loop (i.e. Delta pPORA and Delta pPORB), clones MatDelta A1.1 and MatDelta 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.

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, Delta PORA, and Delta 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.

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-1 antipain and 1 µg ml-1 pepstatin, which efficiently block the POR-degrading stromal protease (39).

Assembly Assay of LHPP-- Different amounts of gel-filtered PORA-Pchlide (ZnPP) b-NADPH, Delta PORA-Pchlide (ZnPP) b-NADPH, PORB-Pchlide (ZnPP) a-NADPH, and Delta 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.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 3alpha ,20beta -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 Delta 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, Delta pPORA, and Delta 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 (Delta pPORA) and precursor PORB (Delta 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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Fig. 2.   Chloroplast import of the various authentic and truncated POR precursors. Chloroplasts were prepared from light-grown barley seedlings and added to equal amounts of radiolabeled (Delta )pPORA and (Delta )pPORB polypeptides, which had been synthesized as described in the legend to Fig. 1. Import was studied at 23 °C in the dark in the presence of 2 mM Mg-ATP in assay mixtures containing 5-ALA dissolved in phosphate buffer or phosphate buffer alone. After 15 min, import was terminated either by adding thermolysin to a 200 µg·ml-1 final concentration (+ Thl) or adding an equal volume of doubly concentrated SDS-sample buffer (- Thl). Assays that had been incubated with thermolysin were supplemented with EGTA prior to final analysis. Protein was resolved by SDS-PAGE. A, the autoradiogram shows precursor and mature POR protein levels in chloroplasts containing (+ 5-ALA) or lacking (- 5-ALA) the exogenous 5-ALA-derived Pchlide. B, as in A but showing mature POR protein levels in thermolysin-treated, repurified chloroplasts after an additional 30-min postimport incubation in darkness. Lanes 1, 6, 11, and 16 show respective input standards. C, as in B but after a 30-min postimport incubation in white light. Note the destabilization of all imported POR proteins in the illuminated samples. In case of the PORB and Delta PORB, an additional processing step occurs, which in either case gives rise to a slightly smaller product, as compared with the freshly imported proteins (A).

In the case of chloroplasts that lacked the 5-ALA-derived Pchlide, no import of the pPORA and its Delta 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 Delta 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 (Delta )POR-pigment-NADPH complexes (22). PORA and Delta PORA were incubated for 15 min in the dark with Pchlide b and NADPH, whereas PORB and Delta 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 (Delta )POR-Pchlide-NADPH ternary complexes were determined in acetone (22).

When the levels of PORA- and Delta PORA-bound Pchlide b were compared, almost no difference was observed; PORA and Delta PORA bound practically indistinguishable amounts of the pigment (Fig. 3A, solid versus dashed line, respectively). Similar results were obtained for the PORB and Delta 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 Delta PORA and Delta PORB polypeptides were enzymatically active.


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Fig. 3.   Pigment binding properties of Delta PORA and Delta PORB. Equal amounts of PORA, Delta PORA, PORB, and Delta PORB polypeptides, which had been synthesized in vitro as described in Fig. 1, were reconstituted to POR-pigment-NADPH complexes and subjected to gel filtration on Sephadex G15. A, fluorescence emission spectra of acetone-extracted PORA-Pchlide b-NADPH (solid line) and Delta PORA-Pchlide b-NADPH (dashed line) complexes were recorded at 23 °C at an excitation wavelength of 440 nm. B, as in A but with PORB-Pchlide a-NADPH (solid line) and Delta PORB-Pchlide a-NADPH (dashed line) complexes. Note that the authentic and truncated POR polypeptides each bind practically identical Pchlide levels.

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 Delta PORA, PORA, Delta 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 Delta PORA and Delta 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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Fig. 4.   Protease sensitivity of reconstituted POR-pigment complexes. POR-Pchlide-NADPH complexes containing equal amounts of the different authentic and truncated POR proteins were prepared as described in Fig. 3 and purified by gel filtration. Parallel samples were then either exposed to white light, to induce enzymatic Chlide formation, or kept in darkness. Subsequently, a stromal protease preparation from barley chloroplasts was added, and the assays were incubated in the dark. After various time intervals, aliquots were withdrawn, and protein was precipitated with trichloroacetic acid, run electrophoretically, detected by autoradiography. As controls, the naked (Delta )PORA and (Delta )PORB proteins, lacking their bound substrates and products, were used. The different curves show the levels of the PORA (A) and Delta PORA (B) as well as PORB (C) and Delta PORB (D) in the respective POR-Pchlide-NADPH (solid lines) and POR-Chlide-NADP+ complexes (dotted lines), as compared with the amount of the naked POR polypeptides (dashed lines). Note that all Pchlide-complexed POR proteins are stable, whereas the respective POR-product complexes undergo rapid degradation. In all cases, this latter decline in POR protein levels is even faster than that of the naked, non-pigment-complexed proteins.

We next asked whether the extra loop might be involved in the formation of higher molecular weight PORA-PORB supracomplexes. Delta PORA-Pchlide b-NADPH, PORA-Pchlide b-NADPH, Delta 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-(Delta )PORA-(Delta )PORB supracomplexes or nonassembled free subunits were pooled and analyzed by SDS-PAGE and autoradiography (13).


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Fig. 5.   Assembly competence of Delta PORA and Delta PORB. POR-pigment-NADPH complexes that had been reconstituted and purified by gel filtration as described in the legend to Fig. 3, were mixed in the indicated combinations and incubated for 15 min in the dark. Then half of the incubation mixtures was precipitated with trichloroacetic acid, whereas the other half was subjected to gel filtration (13). A, autoradiogram showing the different POR polypeptides contained in the various ternary complexes prior to gel filtration. B, autoradiograms showing pooled fractions collected during gel filtration of incubation mixtures containing equimolar amounts of the pigment-complexed Delta PORB and PORA (a), PORB and PORA (b), Delta PORB and Delta PORA (c), and PORB and Delta PORA (d), respectively. Note that only the authentic PORA and PORB polypeptides are able to establish higher molecular weight complexes (panel b, fraction 5), whereas the truncated proteins are not.

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 (Delta )PORA-Pchlide b-NADPH and (Delta )PORB-Pchlide a-NADPH had no impact on this negative result.

    DISCUSSION
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ABSTRACT
INTRODUCTION
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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 beta -sheet that is composed of seven beta -strands (beta -1 to beta -7) surrounded by eight (pea) or nine (Synechocystis) alpha -helices (alpha -A to alpha -I) (44, 45). Just before the highly conserved YXXXK motif, which is located in helix alpha -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 beta -sheet structure, and the fourth is located in residues 371-385 of an alpha -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.

    ACKNOWLEDGEMENT

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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; (Delta )POR, truncated POR; ZnPP, zinc protopheophorbide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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