Interplay of four auxiliary factors is required for the assembly of photosystem I reaction center subcomplex

The photosystem I (PSI) complex consisting of reaction center (RC) subunits, several peripheral subunits and many co-factors, is present in the thylakoid membranes of chloroplasts and cyanobacteria. The assembly of RC subunits (PsaA/B) that bind electron transfer co-factors and antenna pigments is an intricate process, and is mediated by several auxiliary factors such as Ycf3, Y3IP1/CGL59, Ycf4 and Ycf37/PYG7/CGL71. However, their precise molecular mechanisms in RC assembly remain to be addressed. Here we purified four PSI auxiliary factors by affinity chromatography, and characterized co-purified PSI assembly intermediates. We suggest that Ycf3 assists the initial assembly of newly synthesized PsaA/B subunits into an RC subcomplex, while Y3IP1 may be involved in transferring the RC subcomplex from Ycf3 to the Ycf4 module that stabilizes it. CGL71 may form an oligomer that transiently interacts with the PSI RC subcomplex, physically protecting it under oxic conditions until association with the peripheral PSI subunits occurs. Together, our results reveal the interplay among four auxiliary factors required for the stepwise assembly of the PSI RC.     

Y3IP1, a Nucleus-Encoded Thylakoid Protein, Cooperates with the Plastid-Encoded Ycf3 Protein in Photosystem I Assembly of Tobacco and Arabidopsis

The intricate assembly of photosystem I (PSI), a large multiprotein complex in the thylakoid membrane, depends on auxiliary protein factors. One of the essential assembly factors for PSI is encoded by ycf3 (hypothetical chloroplast reading frame number 3) in the chloroplast genome of algae and higher plants. To identify novel factors involved in PSI assembly, we constructed an epitope-tagged version of ycf3 from tobacco (Nicotiana tabacum) and introduced it into the tobacco chloroplast genome by genetic transformation. Immunoaffinity purification of Ycf3 complexes from the transplastomic plants identified a novel nucleus-encoded thylakoid protein, Y3IP1 (for Ycf3-interacting protein 1), that specifically interacts with the Ycf3 protein. Subsequent reverse genetics analysis of Y3IP1 function in tobacco and Arabidopsis thaliana revealed that knockdown of Y3IP1 leads to a specific deficiency in PSI but does not result in loss of Ycf3. Our data indicate that Y3IP1 represents a novel factor for PSI biogenesis that cooperates with the plastid genome-encoded Ycf3 in the assembly of stable PSI units in the thylakoid membrane.     

Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography–tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 × 185 Å; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.     

A Thylakoid Membrane Protein Harboring a DnaJ-type Zinc Finger Domain Is Required for Photosystem I Accumulation in Plants

Photosystem I (PSI) is a large pigment-protein complex and one of the two photosystems that drive electron transfer in oxygenic photosynthesis. We identified a nuclear gene required specifically for the accumulation of PSI in a forward genetic analysis of chloroplast biogenesis in maize. This gene, designated psa2, belongs to the “GreenCut” gene set, a group of genes found in green algae and plants but not in non-photosynthetic organisms. Disruption of the psa2 ortholog in Arabidopsis likewise resulted in the specific loss of PSI proteins. PSA2 harbors a conserved domain found in DnaJ chaperones where it has been shown to form a zinc finger and to have protein-disulfide isomerase activity. Accordingly, PSA2 exhibited protein-disulfide reductase activity in vitro. PSA2 localized to the thylakoid lumen and was found in a ∼250-kDa complex harboring the peripheral PSI protein PsaG but lacking several core PSI subunits. PSA2 mRNA is coexpressed with mRNAs encoding various proteins involved in the biogenesis of the photosynthetic apparatus with peak expression preceding that of genes encoding structural components. PSA2 protein abundance was not decreased in the absence of PSI but was reduced in the absence of the PSI assembly factor Ycf3. These findings suggest that a complex harboring PSA2 and PsaG mediates thiol transactions in the thylakoid lumen that are important for the assembly of PSI.     

The plastid genome-encoded Ycf4 protein functions as a nonessential assembly factor for photosystem I in higher plants

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.     

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1_mod and D2_mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1_mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2_mod contained D2/cytochrome b₅₅₉ with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1_mod but not D2_mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

Analysis of isolated assembly complexes provides new insights into the early stages of photosystem II biogenesis.     

The evolutionarily conserved tetratrico peptide repeat protein pale yellow green7 is required for photosystem I accumulation in Arabidopsis and copurifies with the complex

Pale yellow green7-1 (pyg7-1) is a photosystem I (PSI)-deficient Arabidopsis (Arabidopsis thaliana) mutant. PSI subunits are synthesized in the mutant, but do not assemble into a stable complex. In contrast, light-harvesting antenna proteins of both photosystems accumulate in the mutant. Deletion of Pyg7 results in severely reduced growth rates, alterations in leaf coloration, and plastid ultrastructure. Pyg7 was isolated by map-based cloning and encodes a tetratrico peptide repeat protein with homology to Ycf37 from Synechocystis. The protein is localized in the chloroplast associated with thylakoid membranes and copurifies with PSI. An independent pyg7 T-DNA insertion line, pyg7-2, exhibits the same phenotype. pyg7 gene expression is light regulated. Comparison of the roles of Ycf37 in cyanobacteria and Pyg7 in higher plants suggests that the ancient protein has altered its function during evolution. Whereas the cyanobacterial protein mediates more efficient PSI accumulation, the higher plant protein is absolutely required for complex assembly or maintenance.     

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI). With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Deltaycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Deltaycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.     

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI) [A. Wilde, K. Lünser, F. Ossenbühl, J. Nickelsen, T. Börner, Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content, Biochem. J. 357 (2001) 211-216]. With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Δycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Δycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.     

Characterization of a new gammaTuRC subunit with WD repeats

The gamma-tubulin ring complex (gammaTuRC), consisting of multiple protein subunits, can nucleate microtubule assembly. Although many subunits of the gammaTuRC have been identified, a complete set remains to be defined in any organism. In addition, how the subunits interact with each other to assemble into gammaTuRC remains largely unknown. Here, we report the characterization of a novel gammaTuRC subunit, Drosophila gamma ring protein with WD repeats (Dgp71WD). With the exception of gamma-tubulin, Dgp71WD is the only gammaTuRC component identified to date that does not contain the grip motifs, which are signature sequences conserved in gammaTuRC components. By performing immunoprecipitations after pair-wise coexpression in Sf9 cells, we show that Dgp71WD directly interacts with the grip motif-containing gammaTuRC subunits, Dgrips84, 91, 128, and 163, suggesting that Dgp71WD may play a scaffolding role in gammaTuRC organization. We also show that Dgrips128 and 163, like Dgrips84 and 91, can interact directly with gamma-tubulin. Coexpression of any of these grip motif-containing proteins with gamma-tubulin promotes gamma-tubulin binding to guanine nucleotide. In contrast, in the same assay Dgp71WD interacts with gamma-tubulin but does not facilitate nucleotide binding.     

Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803

The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.     

Role of subunits in eukaryotic Photosystem I.

Photosystem I (PSI) of eukaryotes has a number of features that distinguishes it from PSI of cyanobacteria. In plants, the PSI core has three subunits that are not found in cyanobacterial PSI. The remaining 11 subunits of the core are conserved but several of the subunits have a different role in eukaryotic PSI. A distinguishing feature of eukaryotic PSI is the membrane-imbedded peripheral antenna. Light-harvesting complex I is composed of four different subunits and is specific for PSI. Light-harvesting complex II can be associated with both PSI and PSII. Several of the core subunits interact with the peripheral antenna proteins and are important for proper function of the peripheral antenna. The review describes the role of the different subunits in eukaryotic PSI. The emphasis is on features that are different from cyanobacterial PSI.     

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.     

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.     

A novel zinc finger is required for Mcm10 homocomplex assembly

Mcm10 is a DNA replication factor that interacts with multiple subunits of the MCM2-7 hexameric complex. We report here that Mcm10 self-interacts and assembles into large homocomplexes (approximately 800 kDa). A conserved domain of 210 amino acid residues is sufficient for mediating self-interaction and complex assembly. A novel zinc finger within the conserved domain, CX10CX11CX2H, is essential for the homocomplex formation. Mutant alleles with amino acid substitutions at conserved cysteines and histidine in the zinc finger fail to assemble homocomplexes. A defect in homocomplex assembly correlates with defects in DNA replication and cell growth in the mutants. These observations suggest that homocomplex assembly is essential for Mcm10 function. Multisubunit Mcm10 homocomplexes may provide the structural basis for Mcm10 to interact with multiple subunits of the MCM2-7 hexamer.     

The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27

The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.     

Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes

Protein-protein interaction modules containing so-called tetratricopeptide repeats (TPRs) mediate the assembly of Hsp70/Hsp90 multi-chaperone complexes. The TPR1 and TPR2A domains of the Hsp70/Hsp90 adapter protein p60/Hop specifically bind to short peptides corresponding to the C-terminal tails of Hsp70 and Hsp90, respectively, both of which contain the highly conserved sequence motif EEVD-COOH. Here, we quantitatively assessed the contribution of TPR-mediated peptide recognition to Hsp70.Hop.Hsp90 complex formation. The interaction of TPR2A with the C-terminal pentapeptide of Hsp90 (MEEVD) is identified as the core contact for Hop binding to Hsp90. (In peptide sequences, italics are used to highlight residues specific for Hsp70 or Hsp90.) In contrast, formation of the Hsp70.Hop complex depends not only on recognition of the C-terminal Hsp70 heptapeptide (PTIEEVD) by TPR1 but also on additional contacts between Hsp70 and Hop. The sequence motifs for TPR1 and TPR2A binding were defined by alanine scanning of the C-terminal octapeptides of Hsp70 and Hsp90 and by screening of combinatorial peptide libraries. Asp0 and Val-1 of the EEVD motif are identified as general anchor residues, but the highly conserved glutamates of the EEVD sequence, which are critical in Hsp90 binding by TPR2A, do not contribute appreciably to the interaction of Hsp70 with TPR1. Rather, TPR1 prefers hydrophobic amino acids in these positions. Moreover, the TPR domains display a pronounced tendency to interact preferentially with hydrophobic aliphatic and aromatic side chains in positions -4 and -6 of their respective peptide ligands. Ile-4 in Hsp70 and Met-4 in Hsp90 are most important in determining the specific binding of TPR1 and TPR2A, respectively.     

TRAPP II Complex Assembly Requires Trs33 or Trs65

TRAPP is a multi-subunit complex that acts as a Ypt/Rab activator at the Golgi apparatus. TRAPP exists in two forms: TRAPP I is comprised of five essential and conserved subunits and TRAPP II contains two additional essential and conserved subunits, Trs120 and Trs130. Previously, we have shown that Trs65, a nonessential fungi-specific TRAPP subunit, plays a role in TRAPP II assembly. TRS33 encodes another nonessential but conserved TRAPP subunit whose function is not known. Here, we show that one of these two subunits, nonessential individually, is required for TRAPP II assembly. Trs33 and Trs65 share sequence, intracellular localization and interaction similarities. Specifically, Trs33 interacts genetically with both Trs120 and Trs130 and physically with Trs120. In addition, trs33 mutant cells contain lower levels of TRAPP II and exhibit aberrant localization of the Golgi Ypts. Together, our results indicate that in yeast, TRAPP II assembly is an essential process that can be accomplished by either of two related TRAPP subunits. Moreover, because humans express two Trs33 homologues, we propose that the requirement of Trs33 for TRAPP II assembly is conserved from yeast to humans.     

Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex

Eleven genes (ndhA-ndhK) encoding proteins homologous to the subunits of bacterial and mitochondrial NADH dehydrogenase (complex I) were found in the plastid genome of most land plants. These genes encode subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in photosystem I (PSI) cyclic electron transport and chlororespiration. Although the chloroplast NDH is believed to be closely and functionally related to the cyanobacterial NDH-1L complex, extensive proteomic, genetic and bioinformatic studies have discovered many novel subunits that are specific to higher plants. On the basis of extensive mutant characterization, the chloroplast NDH complex is divided into four parts, the A, B, membrane and lumen subcomplexes, of which subunits in the B and lumen subcomplexes are specific to higher plants. These results suggest that the structure of NDH has been drastically altered during the evolution of land plants. Furthermore, chloroplast NDH interacts with multiple copies of PSI to form the unique NDH-PSI supercomplex. Two minor light-harvesting-complex I (LHCI) proteins, Lhca5 and Lhca6, are required for the specific interaction between NDH and PSI. The evolution of chloroplast NDH in land plants may be required for development of the function of NDH to alleviate oxidative stress in chloroplasts. In this review, we summarize recent progress on the subunit composition and structure of the chloroplast NDH complex, as well as the information on some factors involved in its assembly. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

The stable assembly of newly synthesized PsaE into the photosystem I complex occurring via the exchange mechanism is facilitated by electrostatic interactions

Photosystem I (PSI) is a photochemically active membrane protein complex that functions at the reducing site of the photosynthetic electron-transfer chain as plastocyanin-ferredoxin oxidoreductase. PsaE, a peripheral subunit of the PSI complex, plays an important role in the function of PSI. PsaE is involved in the docking of ferredoxin/flavodoxin to the PSI complex and also participates in the cyclic electron transfer around PSI. The molecular characterization of the assembly of newly synthesized PsaE in the thylakoid membranes or in isolated PSI complexes is the subject of the present study. For this purpose the Mastigocladus laminosus psaE gene was cloned and overexpressed in Escherichia coli, and the resulting PsaE protein was purified to homogeneity by affinity chromatography. The purified PsaE was then introduced into thylakoids isolated from M. laminosus, and the newly introduced PsaE subunit saturates the membrane. The solubilization and separation of the different thylakoid protein complexes indicated that PsaE accumulates specifically in its functional location, the PSI complex. A similar stable assembly was detected when PsaE was introduced into purified PSI complexes, i.e., in the absence of other thylakoid components. This strongly indicates that the information for the stable assembly of PsaE into PSI lies within the polypeptide itself and within other subunits of the PSI complex that interact with it. To determine the nature of these interactions, the assembly reaction was performed in conditions affecting the ionic/osmotic strength. We found that altering the ionic strength significantly affects the capability of PsaE to assemble into isolated thylakoids or PSI complexes, strongly supporting the fact that electrostatic interactions are formed between PsaE and other PSI subunits. Moreover, the data suggest that the formation of electrostatic interactions occurs concomitantly with an exchange step in which newly introduced PsaE replaces the subunit present in situ.