Characterization of a highly purified, fully active, crystallizable RC-LH1-PufX core complex from Rhodobacter sphaeroides.

Photosynthetic complexes in bacteria absorb light and undergo photochemistry with high quantum efficiency. We describe the isolation of a highly purified, active, reaction center-light-harvesting 1-PufX complex (RC-LH1-PufX core complex) from a strain of the photosynthetic bacterium, Rhodobacter sphaeroides, which lacks the light-harvesting 2 (LH2) and contains a 6 histidine tag on the H subunit of the RC. The complex was solubilized with diheptanoyl-sn-glycero-3-phosphocholine (DHPC), and purified by Ni-affinity, size-exclusion and ion-exchange chromatography in dodecyl maltoside. SDS-PAGE analysis shows the complex to be highly purified. The quantum efficiency was determined by measuring the charge separation (DQA --> D+QA -) in the RC as a function of light intensity. The RC-LH1-PufX complex had a quantum efficiency of 0.95 +/- 0.05, indicating full activity. The stoichiometry of LH1 subunits per RC was determined by two independent methods: (i) solvent extraction and absorbance spectroscopy of bacteriochlorophyll, and (ii) density scanning of the SDS-PAGE bands. The average stoichiometry from the two measurements was 13.3 +/- 0.9 LH1/RC. The presence of PufX was observed in SDS-PAGE gels at a stoichiometry of 1.1 +/- 0.1/RC. Crystals of the core complex have been obtained which diffract X-rays to 12 A. A preliminary analysis of the space group and unit cell analysis indicated a P1 space group with unit cell dimensions of a = 76.3 A, b = 137.2 A, c = 137.5 A; alpha = 60.0 degrees , beta = 89.95 degrees , gamma =90.02 degrees .

The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism.

PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex. To unravel the mechanism by which this assembly takes place, the following steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex. Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used. The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI. The latter replaces the PsaD subunit that is present in situ within the complex. In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes.

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus.

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.

The precursor of PsaD assembles into the photosystem I complex in two steps.

The present study addresses the assembly in the chloroplast thylakoid membranes of PsaD, a peripheral membrane protein of the photosystem I complex. Located on the stromal side of the thylakoids, PsaD was found to assemble in vitro into the membranes in its precursor (pre-PsaD) and also in its mature (PsaD) form. Newly assembled unprocessed pre-PsaD was resistant to NaBr and alkaline wash. Yet it was sensitive to proteolytic digestion. In contradistinction, when the assembled precursor was processed, the resulting mature PsaD was resistant to proteases to the same extent as endogenous [correction of endogeneous] PsaD. The accumulation of protease-resistant PsaD in the thylakoids correlated with the increase of mature-PsaD in the membranes. This protection of mature PsaD from proteolysis could not be observed when PsaD was in a soluble form-i.e. not assembled within the thylakoids. The data suggest that pre-PsaD assembles to the membranes and only in a second step processing takes place. The observation that the assembly of pre-PsaD is affected by salts to a much lesser extent than that of mature-PsaD supports a two-step assembly of pre-PsaD.

Function and organization of Photosystem I polypeptides.

Photosystem I functions as a plastocyanin:ferredoxin oxidoreductase in the thylakoid membranes of chloroplasts and cyanobacteria. The PS I complex contains the photosynthetic pigments, the reaction center P700, and five electron transfer centers (A0, A1, FX, FA, and FB) that are bound to the PsaA, PsaB, and PsaC proteins. In addition, PS I complex contains at least eight other polypeptides that are accessory in their functions. Recent use of cyanobacterial molecular genetics has revealed functions of the accessory subunits of PS I. Site-directed mutagenesis is now being used to explore structure-function relations in PS I. The overall architecture of PSI complex has been revealed by X-ray crystallography, electron microscopy, and biochemical methods. The information obtained by different techniques can be used to propose a model for the organization of PS I. Spectroscopic and molecular genetic techniques have deciphered interaction of PS I proteins with the soluble electron transfer partners. This review focuses on the recent structural, biochemical and molecular genetic studies that decipher topology and functions of PS I proteins, and their interactions with soluble electron carriers.

The carboxyl-terminal region of the spinach PsaD subunit contains information for its specific assembly into plant thylakoids.

The assembly of the multi-subunit membrane-protein Photosystem I (PS I) complex involves incorporation of peripheral proteins into the complex. Here we studied assembly of the PsaD subunit of the cyanobacterial and plant PS I into the thylakoid membranes. We generated partial and chimeric psaD genes from which labeled proteins were synthesized in vitro. Assembly of these proteins into the cyanobacterial or plant thylakoids was assayed. The deletion of leader sequence and N-terminal extension of spinach prePsaD did not inhibit its assembly into spinach or cyanobacterial thylakoids. Addition of these sequences to the cyanobacterial PsaD did not enable it to assemble into plant thylakoids. Moreover, these additions significantly decreased the ability of the chimeric proteins to assemble into cyanobacterial thylakoids. In contrast, when the carboxyl-terminal half of cyanobacterial PsaD was replaced by the corresponding region of the spinach PsaD, the chimeric protein could assemble into both spinach and cyanobacterial thylakoids. Therefore, information in the carboxyl-terminal region of spinach PsaD is crucial for its assembly into plant thylakoids.

Assembly of the chlorophyll-protein complexes.

The biogenesis of photosynthetic complexes in plants and algae is a multi-step process that involves intricate coordination of steps in two intracellular compartments, the chloroplast and the cytoplasm. The process initiates with the transcription and translation of the various polypeptide subunits. The nuclear-encoded Chl-binding proteins are translated on cytoplasmic ribosomes as precursors that have a transit (leader) sequence at their amino-terminus. The precursors are post-translationally imported into the chloroplasts, proteolytically processed into their mature forms, inserted into the thylationally imported into the chloroplasts, proteolytically processed into their mature forms, inserted into the thylakoid membrane, and bound to their co-factors (and pigments) and with other subunits to form an active complex. The order and mechanisms by which these events occur, are currently being discovered. Electrostatic interactions, the 'positive inside rule', interhelix interactions, interactions with lipids and chaperone proteins affect the insertion and stabilization of the Chl-proteins in the thylakoids. This review describes the events occurring during the integration and organization of the Chl-proteins.

Nucleotide sequence of the psbE, psbF and trnM genes from the chloroplast genome of Chlamydomonas reinhardtii.

We have determined the nucleotide sequences of the psbE and psbF genes, which encode the alpha and beta subunits, respectively, of cytochrome b-559, from the chloroplast genome of the green alga Chlamydomonas reinhardtii. In contrast to other organisms psbE is not co-transcribed with psbF. The primary structures of the gene products are very similar to the equivalent proteins in cyanobacteria and plants. Each subunit contains a single histidine residue that is thought to ligate haem. Upstream of the psbE gene, a trnM gene is located which encodes an elongator tRNA(Met) molecule.

Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I.

We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.

High levels of photosystem I subunit II (PsaD) mRNA result in the accumulation of the PsaD polypeptide only in the presence of light.

The light-regulated mRNA and polypeptide accumulation of the nuclear encoded subunit II (PsaD) of the photosystem I reaction center was studied during the greening of etiolated spinach seedlings. Upon exposure to continuous white light, the mRNA, detected at low levels in etiolated seedlings, accumulated in a specific pattern. In contrast, the PsaD subunit could not be detected in the etiolated seedlings; the polypeptide could first be detected in thylakoid membranes approximately 4 h after exposure to continuous light. A pulse of red light induced the expression of the PsaD mRNA, but the polypeptide could not be detected unless the seedlings were exposed to light. In the light (but not in the dark), the PsaD mRNA was found associated with the polysomal fraction. Taken together, the data suggest a dual regulatory mechanism in which both the level of mRNA and the presence of light control the accumulation of the PsaD polypeptide.

Subunit III (Psa-F) of photosystem I reaction center of the C4 dicotyledon Flaveria trinervia.

An immunological survey of C3, C4 and C3-C4-intermediate Flaveria species showed that subunit III (PsaF) of the photosystem I reaction center (PSI-RC) is present in all these species. This was confirmed by the isolation of the gene encoding the PSI-RC subunit III (PsaF) from Flaveria trinervia, the first psaF gene to be isolated from a C4 plant. The deduced amino acid sequence showed a high degree of similarity to the corresponding protein of spinach which is a C3 species. A region of 17 hydrophobic amino acids in the C-terminal part of the F. trinervia protein was found to be especially conserved in all PsaF proteins studied so far (cyanobacteria and Chlamydomonas).

Accumulation of a light-harvesting chlorophyll a/b protein in the chloroplast grana lamellae. The lateral migration of the membrane protein precursor is independent of its processing.

The events that follow the import of pLHCPIIb, the apoprotein precursor of the major light-harvesting complex of photosystem II, were studied in intact pea chloroplasts. The distribution of the events of insertion into the membrane, and processing, to yield the mature form (LHCP) between stromal and granal lamellae regions of the thylakoids were followed. pLHCP was preferentially inserted into stromal lamellae (SL) from which it migrated to granal lamellae (GL). Migration occurred before or after processing, suggesting that migration and processing are independent of each other. When migration was slowed down, LHCP accumulated in SL. Prolonged inhibition of migration induced degradation of LHCP that had accumulated in SL, whereas inhibition of processing did not affect the migration of pLHCP into GL. A small difference in electrophoretic mobility was noted between LHCP in SL and in GL. The predominant mature form in SL migrated more slowly than LHCP from GL. When thylakoids were subjected to trypsin, all of the LHCP embedded in SL underwent cleavage, whereas up to 60% of the radioactive LHCP in GL was resistant to the enzyme. The possible implications of the differences in size and in the sensitivity to trypsin of LHCP are discussed.

Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane.

Molecular chaperones, including those belonging to the 70-kDa family of heat shock proteins (HSP70), assist both the translocation of proteins across membranes and their assembly into oligomeric complexes. We purified a chloroplast HSP70 (ct-HSP70) and demonstrated that it plays a major role in the insertion of the precursor of the major light-harvesting complex of photosystem II (pLHCP; an integral membrane protein) into the thylakoids (the inner membranes of the chloroplast). Addition of the purified ct-HSP70 is necessary for efficient insertion of pLHCP into isolated thylakoid membranes. This activity of the purified ct-HSP70 is similar to that previously reported for the total stromal extract. When the chloroplast stromal extract is depleted of HSP70, a correlative reduction in the insertion activity of pLHCP is observed. The interaction between the ct-HSP70 and pLHCP involves physical association. The purified HSP70 acts directly on the membrane protein, presumably prevents its refolding, and thereby helps to maintain its competence for insertion into membranes.

Assembly and processing of subunit II (PsaD) precursor in the isolated photosystem-I complex.

The precursor of photosystem I (PSI) subunit II (pre-subunit II) synthesized in vitro, was found to bind to the holo-PSI complex, both within the thylakoids and outside, after detergent extraction of PSI from the membranes. Chloroplast stromal fraction added to the purified PSI complexes, containing the labeled pre-subunit II, induced the processing of the precursor to the mature form. This implies that processing can occur within the isolated complex, after the integration of the precursor. The results presented suggest that certain aspects of biogenesis of membranal protein complexes can be studied in detergent-extracted purified complexes.

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.

The composition and organization of photosystem I.

Photosystem I, extensively studied in the past decade, was shown to be homologous in all photosynthetic organisms of the higher plants type. Its core complex was found to be highly conserved through evolution from cyanobacteria to higher plants. The genes coding for the subunits of CCI were isolated and the resulting sequences provided information about secondary structural elements. These suggested secondary structures enabled the prediction of the topology of these subunits in the photosynthetic membrane. Structural studies using both electron microscopy and X-ray crystallography, on isolated particles as well as on the complexes in the photosynthetic membrane, led to a better understanding of the overall structure of CCI. Recently two forms of three dimensional crystals of CCI were obtained. These crystals contain all the original components of CCI (both protein and pigments); these components have not been altered by crystallization. It is expected that a detailed crystallographic analysis of these crystals, together with biochemical, spectroscopical and molecular biology studies, will eventually lead to the elucidation of the high resolution structure of the photosystem I core complex and to the understanding of the exact role and mode of action of this complex in the photosynthetic membrane.

On some of the in organello processes involved in the biogenesis of chlorophyll-protein complexes.

The biogenesis and assembly of chlorophyll-protein complexes consist of many steps. These are initiated with the transcription and translation of the different polypeptide components constituting the complexes. For the nuclear-encoded subunits the synthesis takes place in the cytoplasm, and they are synthesized as precursors, which are later imported into the chloroplast. Within the organelle, the precursors are inserted into the thylakoid membranes, as well as being processed to their mature forms. The different nuclear- and chloroplast-encoded subunits assemble together, and bind the pigments and other cofactors to form the active pigmented-complex. In the present article, we discuss only the in organello processes of the biogenesis. We describe the pathways taken by two nuclear-encoded thylakoid proteins, the precursor of the main light-harvesting chlorophyll-protein of photosystem II (pLHCP) and the precursor of photosystem I subunit II (pre subunit II). These polypeptide subunits, that are located in two different photosynthetic complexes, differ from each other. While pLHCP is an integral membrane protein, which binds pigments, photosystem I-subunit II is a peripheral membrane protein, located on the stromal side of the thylakoids, and is not predicted to span it. The differences and the common features of the in organello biogenesis pathways of these two proteins are discussed.

Monomeric and trimeric forms of photosystem I reaction center of Mastigocladus laminosus: crystallization and preliminary characterization.

Photosystem I (PSI) reaction centers (RCs) of the thermophilic cyanobacterium Mastigocladus laminosus were purified and characterized. The PSI RC was obtained in two forms, monomeric and trimeric. The two forms contained the same number of pigments per P700 and displayed similar photochemical activities. The two forms had nearly identical polypeptide subunit compositions; the only observed difference was an additional subunit of about 12 kDa observed in the trimeric form. The purified preparations of both the monomeric and the trimeric forms were used for crystallization and preliminary crystallographic analysis. The trimeric PSI RC preparations produced several three-dimensional crystal forms, one of which, the "hexagonal needle" form (THN), had a hexagonal unit cell with dimensions of 300 x 300 x 160 A, containing four PSI RC trimers. The monomeric preparations also produced single crystals of several forms under various crystallization conditions. One of these crystal forms, the "hexagonal plate" (MHP), diffracted to a resolution of about 5.5 A. It had a hexagonal unit cell with dimensions of 192 x 192 x 163 A, containing six PSI RC monomers. Comparison of the PSI RCs in the crystals with those in the precrystallization preparations demonstrated that neither the monomeric nor the trimeric form of PSI RC was altered by the crystallization process. Both forms retained their original polypeptide subunit composition and their pigment content.