ABSTRACT
As an essential intrinsic component of photosystem II (PSII) in all oxygenic photosynthetic organisms, heme-bridged heterodimer cytochrome b559 (Cyt b559) plays critical roles in protection and assembly of PSII. However, the underlying mechanisms of Cyt b559 assembly are largely unclear. Here, we characterized the Arabidopsis (Arabidopsis thaliana) rph1 (resistance to Phytophthora1) mutant, which was previously shown to be susceptible to the oomycete pathogen Phytophthora brassicae. Loss of RPH1 leads to a drastic reduction in PSII accumulation, which can be primarily attributed to the defective formation of Cyt b559. Spectroscopic analyses showed that the heme level in PSII supercomplexes isolated from rph1 is significantly reduced, suggesting that RPH1 facilitates proper heme assembly in Cyt b559. Due to the loss of RPH1-mediated processes, a covalently bound PsbE-PsbF heterodimer is formed during the biogenesis of PSII. In addition, rph1 is highly photosensitive and accumulates elevated levels of ROS under photoinhibitory light conditions. RPH1 is a conserved intrinsic thylakoid protein present in green algae and terrestrial plants, but absent in Synechocystis, and it directly interacts with the subunits of Cyt b559. Thus, our data demonstrate that RPH1 represents a chloroplast acquisition specifically promoting the efficient assembly of Cyt b559, probably by mediating proper heme insertion into the apo-Cyt b559 during the initial phase of PSII biogenesis.
ABSTRACT
F-type ATP synthases produce nearly all of the ATP found in cells. The catalytic module F1 commonly comprises an α3ß3 hexamer surrounding a γ/ε stalk. However, it is unclear how these subunits assemble to form a catalytic motor. In this work, we identified and characterized a chloroplast protein that interacts with the CF1ß, γ, and ε subunits of the chloroplast ATP synthase and is required for assembly of its F1 module. We named this protein BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE1 (BFA1) and determined its crystal structure at 2.8-Å resolution. BFA1 is comprised primarily of two interacting ß-barrels that are oriented nearly perpendicularly to each other. The contact region between BFA1 and the CF1ß and γ subunits was further mapped by yeast two-hybrid assays. An in silico molecular docking analysis was performed and revealed close fitting contact sites without steric conflicts between BFA1 and CF1ß/γ. We propose that BFA1 acts mainly as a scaffold protein promoting the association of a CF1α/ß heterodimer with CF1γ. The subsequent assembly of other CF1α/ß heterodimers may shift the position of the CF1γ subunit to complete assembly of the CF1 module. This CF1 assembly process is likely to be valid for other F-type ATP synthases, as their structures are highly conserved.
Subject(s)
Cell Nucleus/metabolism , Chloroplast Proton-Translocating ATPases/metabolism , Chloroplasts/metabolism , Cell Nucleus/genetics , Chloroplast Proton-Translocating ATPases/genetics , Chloroplasts/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Two-Hybrid System TechniquesABSTRACT
Photosystem II (PSII), a multisubunit protein complex of the photosynthetic electron transport chain, functions as a water-plastoquinone oxidoreductase, which is vital to the initiation of photosynthesis and electron transport. Although the structure, composition, and function of PSII are well understood, the mechanism of PSII biogenesis remains largely elusive. Here, we identified a nuclear-encoded pentatricopeptide repeat (PPR) protein LOW PHOTOSYNTHETIC EFFICIENCY 1 (LPE1; encoded by At3g46610) in Arabidopsis, which plays a crucial role in PSII biogenesis. LPE1 is exclusively targeted to chloroplasts and directly binds to the 5' UTR of psbA mRNA which encodes the PSII reaction center protein D1. The loss of LPE1 results in less efficient loading of ribosome on the psbA mRNA and great synthesis defects in D1 protein. We further found that LPE1 interacts with a known regulator of psbA mRNA translation HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) and facilitates the association of HCF173 with psbA mRNA. More interestingly, our results indicate that LPE1 associates with psbA mRNA in a light-dependent manner through a redox-based mechanism. This study enhances our understanding of the mechanism of light-regulated D1 synthesis, providing important insight into PSII biogenesis and the functional maintenance of efficient photosynthesis in higher plants.
Subject(s)
Arabidopsis Proteins/biosynthesis , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Eukaryotic Initiation Factors/metabolism , Gene Expression Regulation, Plant , Light , Membrane Transport Proteins/metabolism , Photosystem II Protein Complex/biosynthesis , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Eukaryotic Initiation Factors/genetics , Membrane Transport Proteins/genetics , Photosystem II Protein Complex/geneticsABSTRACT
The reaction center (RC) of photosystem II (PSII), which is composed of D1, D2, PsbI, and cytochrome b559 subunits, forms at an early stage of PSII biogenesis. However, it is largely unclear how these components assemble to form a functional unit. In this work, we show that synthesis of the PSII core proteins D1/D2 and formation of the PSII RC is blocked specifically in the absence of ONE-HELIX PROTEIN1 (OHP1) and OHP2 proteins in Arabidopsis (Arabidopsis thaliana), indicating that OHP1 and OHP2 are essential for the formation of the PSII RC. Mutagenesis of the chlorophyll-binding residues in OHP proteins impairs their function and/or stability, suggesting that they may function in the binding of chlorophyll in vivo. We further show that OHP1, OHP2, and HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244), together with D1, D2, PsbI, and cytochrome b559, form a complex. We designated this complex the PSII RC-like complex to distinguish it from the RC subcomplex in the intact PSII complex. Our data imply that OHP1, OHP2, and HCF244 are present in this PSII RC-like complex for a limited time at an early stage of PSII de novo assembly and of PSII repair under high-light conditions. In a subsequent stage of PSII biogenesis, OHP1, OHP2, and HCF244 are released from the PSII RC-like complex and replaced by the other PSII subunits. Together with previous reports on the cyanobacterium Synechocystis, our results demonstrate that the process of PSII RC assembly is highly conserved among photosynthetic species.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/metabolism , Chlorophyll Binding Proteins/physiology , Eukaryotic Initiation Factors/physiology , Photosystem II Protein Complex/physiology , Amino Acid Sequence , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chlorophyll Binding Proteins/genetics , Chlorophyll Binding Proteins/metabolism , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Photosynthesis , Photosystem II Protein Complex/metabolism , Sequence Alignment , Thylakoids/metabolismABSTRACT
Using a genetic approach, we have identified and characterized a novel protein, named Msf1 (Maintenance factor for photosystem I), that is required for the maintenance of specific components of the photosynthetic apparatus in the green alga Chlamydomonas reinhardtii Msf1 belongs to the superfamily of light-harvesting complex proteins with three transmembrane domains and consensus chlorophyll-binding sites. Loss of Msf1 leads to reduced accumulation of photosystem I and chlorophyll-binding proteins/complexes. Msf1is a component of a thylakoid complex containing key enzymes of the tetrapyrrole biosynthetic pathway, thus revealing a possible link between Msf1 and chlorophyll biosynthesis. Protein interaction assays and greening experiments demonstrate that Msf1 interacts with Copper target homolog1 (CHL27B) and accumulates concomitantly with chlorophyll in Chlamydomonas, implying that chlorophyll stabilizes Msf1. Contrary to other light-harvesting complex-like genes, the expression of Msf1 is not stimulated by high-light stress, but its protein level increases significantly under heat shock, iron and copper limitation, as well as in stationary cells. Based on these results, we propose that Msf1 is required for the maintenance of photosystem I and specific protein-chlorophyll complexes especially under certain stress conditions.
Subject(s)
Chlamydomonas/metabolism , Chlamydomonas/physiology , Light-Harvesting Protein Complexes/metabolism , Photosynthesis , Plant Proteins/metabolism , Amino Acid Sequence , Biosynthetic Pathways , Chlamydomonas/growth & development , Chlorophyll/metabolism , Genetic Complementation Test , Heat-Shock Response , Light-Harvesting Protein Complexes/chemistry , Mutation/genetics , Photosystem I Protein Complex/metabolism , Photosystem II Protein Complex/metabolism , Plant Proteins/chemistry , Protein Binding , Protein Subunits/metabolism , Stress, Physiological , Thylakoids/metabolismABSTRACT
Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3ß3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1ß subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1ß. Several residues highly conserved in eukaryotic CF1ß are crucial for the BFA3-CF1ß interaction, suggesting a coevolutionary relationship between BFA3 and CF1ß. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1ß at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase.
Subject(s)
Arabidopsis Proteins/metabolism , Chloroplast Proton-Translocating ATPases/metabolism , Arabidopsis/enzymology , Arabidopsis Proteins/genetics , Membrane Proteins/metabolism , Mutation/genetics , Phenotype , Photosynthesis , Protein Binding , Protein Interaction Mapping , Protein Subunits/metabolism , Thylakoids/metabolismABSTRACT
RNA editing is a posttranscriptional process that covalently alters the sequence of RNA molecules and plays important biological roles in both animals and land plants. In flowering plants, RNA editing converts specific cytidine residues to uridine in both plastid and mitochondrial transcripts. Previous studies identified pentatricopeptide repeat (PPR) motif-containing proteins as site-specific recognition factors for cytidine targets in RNA sequences. However, the regulatory mechanism underlying RNA editing was largely unknown. Here, we report that protoporphyrinogen IX oxidase 1 (PPO1), an enzyme that catalyzes protoporphyrinogen IX into protoporphyrin IX in the tetrapyrrole biosynthetic pathway, plays an unexpected role in editing multiple sites of plastid RNA transcripts, most of which encode subunits of the NADH dehydrogenase-like complex (NDH), in the reference plant Arabidopsis thaliana. We identified multiple organellar RNA editing factors (MORFs), including MORF2, MORF8, and MORF9, that interact with PPO1. We found that two conserved motifs within the 22-aa region at the N terminus of PPO1 are essential for its interaction with MORFs, its RNA editing function, and subsequently, its effect on NDH activity. However, transgenic plants lacking key domains for the tetrapyrrole biosynthetic activity of PPO1 exhibit normal RNA editing. Furthermore, MORF2 and MORF9 interact with three PPRs or related proteins required for editing of ndhB and ndhD sites. These results reveal that the tetrapyrrole biosynthetic enzyme PPO1 is required for plastid RNA editing, acting as a regulator that promotes the stability of MORF proteins through physical interaction.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Plastids/enzymology , Plastids/genetics , Protoporphyrinogen Oxidase/metabolism , RNA Editing/genetics , Tetrapyrroles/biosynthesis , Arabidopsis Proteins/genetics , Base Sequence , Chlorophyll/biosynthesis , Flavin-Adenine Dinucleotide/metabolism , Molecular Sequence Data , NADH Dehydrogenase/metabolism , Phenotype , Protein Binding , Protoporphyrinogen Oxidase/genetics , Seedlings/growth & development , Substrate SpecificityABSTRACT
The chloroplast NADH dehydrogenase-like (NDH) complex is involved in cyclic electron transport around photosystem I (PSI) and chlororespiration. Although the NDH complex was discovered more than 20 years ago, its low abundance and fragile nature render it recalcitrant to analysis, and it is thought that some of its subunits remain to be identified. Here, we identified the NDH subunit NdhV that readily disassociates from the NDH complex in the presence of detergent, salt and alkaline solutions. The Arabidopsis ndhv mutant is partially defective in the accumulation of NDH subcomplex A (SubA) and SubE, resulting in impaired NDH activity. NdhV was mainly detected in the wild-type thylakoid membrane, and its accumulation in thylakoids strictly depended on the presence of the NDH complex. Quantitative immunoblot analysis revealed that NdhV and NdhN occur at close to equimolar concentrations. Furthermore, several NDH subunits were co-immunopurified with NdhV using a combination of chemical crosslinking and an affinity chromatography assay. These data indicate that NdhV is an intrinsic subunit of NDH. We found that NdhV did not directly affect NDH activity, but that NDH SubA and SubE were more rapidly degraded in ndhv than in the wild type under high-light treatment. We propose that NdhV is an NDH subunit that stabilizes this complex, especially under high-light conditions.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/metabolism , Chloroplasts/enzymology , NADH Dehydrogenase/metabolism , Arabidopsis Proteins/genetics , Chloroplasts/metabolism , NADH Dehydrogenase/geneticsABSTRACT
In vascular plants, the chloroplast NADH dehydrogenase-like (NDH) complex, a homolog of respiratory NADH:quinone oxidoreductase (Complex I), mediates plastoquinone reduction using ferredoxin as an electron donor in cyclic electron transport around PSI in the thylakoid membrane. In angiosperms, chloroplast NDH is composed of five subcomplexes and forms a supercomplex with PSI. The modular assembly of stroma-protruded subcomplex A, which corresponds to the Q module of Complex I, was recently reported. However, the factors involved in the specific assembly steps have not been completely identified. Here, we isolated an Arabidopsis mutant, chlororespiratory reduction 9 (crr9), defective in NDH activity. The CRR9 gene encodes a novel stromal protein without any known functional domains or motifs. CRR9 is highly conserved in cyanobacteria and land plants but not in green algae, which do not have chloroplast NDH. Blue native-PAGE and immunoblot analyses of thylakoid proteins indicated that formation of subcomplex A was impaired in crr9 CRR9 was specifically required for the accumulation of NdhK, a subcomplex A subunit, in NDH assembly intermediates in the stroma. Furthermore, two-dimensional clear native/SDS-PAGE analysis of the stroma fraction indicated that incorporation of NdhM into NDH assembly intermediate complex 400 was impaired in crr9 These results suggest that CRR9 is a novel factor required for the formation of NDH subcomplex A.
Subject(s)
Arabidopsis Proteins/metabolism , Chloroplast Proteins/metabolism , Chloroplasts/enzymology , Multienzyme Complexes/metabolism , NADH Dehydrogenase/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Conserved Sequence , Cyanobacteria/genetics , Genes, Plant , Mutation/genetics , Protein Subunits/metabolism , Thylakoids/metabolismABSTRACT
Compared with small heat shock proteins (sHSPs) in other organisms, those in plants are the most abundant and diverse. However, the molecular mechanisms by which sHSPs are involved in cell protection remain unknown. Here, we characterized the role of HSP21, a plastid nucleoid-localized sHSP, in chloroplast development under heat stress. We show that an Arabidopsis thaliana knockout mutant of HSP21 had an ivory phenotype under heat stress. Quantitative real-time RT-PCR, run-on transcription, RNA gel blot, and polysome association analyses demonstrated that HSP21 is involved in plastid-encoded RNA polymerase (PEP)-dependent transcription. We found that the plastid nucleoid protein pTAC5 was an HSP21 target. pTAC5 has a C4-type zinc finger similar to that of Escherichia coli DnaJ and zinc-dependent disulfide isomerase activity. Reduction of pTAC5 expression by RNA interference led to similar phenotypic effects as observed in hsp21. HSP21 and pTAC5 formed a complex that was associated mainly with the PEP complex. HSP21 and pTAC5 were associated with the PEP complex not only during transcription initiation, but also during elongation and termination. Our results suggest that HSP21 and pTAC5 are required for chloroplast development under heat stress by maintaining PEP function.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Carrier Proteins/metabolism , Chloroplasts/metabolism , Heat-Shock Proteins/metabolism , Heat-Shock Response , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/chemistry , Carrier Proteins/chemistry , Chloroplasts/drug effects , Chloroplasts/genetics , Chloroplasts/radiation effects , DNA, Chloroplast/metabolism , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Genes, Plant/genetics , Heat-Shock Proteins/chemistry , Heat-Shock Response/drug effects , Heat-Shock Response/genetics , Heat-Shock Response/radiation effects , Light , Mutation/genetics , Phenotype , Plants, Genetically Modified , Protein Binding/drug effects , Protein Binding/radiation effects , Protein Disulfide-Isomerases/metabolism , Protein Structure, Tertiary , Protein Transport/drug effects , Protein Transport/radiation effects , Seedlings/drug effects , Seedlings/growth & development , Seedlings/metabolism , Seedlings/radiation effects , Sequence Deletion , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Subcellular Fractions/radiation effects , Zinc/pharmacologyABSTRACT
During photosynthesis, photosynthetic electron transport generates a proton motive force (pmf) across the thylakoid membrane, which is used for ATP biosynthesis via ATP synthase in the chloroplast. The pmf is composed of an electric potential (ΔΨ) and an osmotic component (ΔpH). Partitioning between these components in chloroplasts is strictly regulated in response to fluctuating environments. However, our knowledge of the molecular mechanisms that regulate pmf partitioning is limited. Here, we report a bestrophin-like protein (AtBest), which is critical for pmf partitioning. While the ΔpH component was slightly reduced in atbest, the ΔΨ component was much greater in this mutant than in the wild type, resulting in less efficient activation of nonphotochemical quenching (NPQ) upon both illumination and a shift from low light to high light. Although no visible phenotype was observed in the atbest mutant in the greenhouse, this mutant exhibited stronger photoinhibition than the wild type when grown in the field. AtBest belongs to the bestrophin family proteins, which are believed to function as chloride (Cl- ) channels. Thus, our findings reveal an important Cl- channel required for ion transport and homeostasis across the thylakoid membrane in higher plants. These processes are essential for fine-tuning photosynthesis under fluctuating environmental conditions.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Thylakoids/metabolism , Hydrogen-Ion Concentration , Photosynthesis/physiology , Proton-Motive Force/physiologyABSTRACT
We have identified hpm91, a Chlamydomonas mutant lacking Proton Gradient Regulation5 (PGR5) capable of producing hydrogen (H2 ) for 25 days with more than 30-fold yield increase compared to wild type. Thus, hpm91 displays a higher capacity of H2 production than a previously characterized pgr5 mutant. Physiological and biochemical characterization of hpm91 reveal that the prolonged H2 production is due to enhanced stability of PSII, which correlates with increased reactive oxygen species (ROS) scavenging capacity during sulfur deprivation. This anti-ROS response appears to protect the photosynthetic electron transport chain from photo-oxidative damage and thereby ensures electron supply to the hydrogenase.
Subject(s)
Algal Proteins/metabolism , Chlamydomonas/metabolism , Hydrogen/metabolism , Protons , Reactive Oxygen Species/metabolism , Genetic Complementation Test , Photochemical ProcessesABSTRACT
Chloroplast NADH dehydrogenase-like complex (NDH) mediates photosystem I cyclic electron transport and chlororespiration in thylakoids. Recently, substantial progress has been made in understanding the structure of NDH, but our knowledge of its assembly has been limited. In this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of NDH (subcomplex A). In addition to further characterization of the previously identified CHLORORESPIRATORY REDUCTION1 (CRR1), CRR6, and CRR7, two novel stromal proteins, CRR41 and CRR42, were discovered. Arabidopsis thaliana mutants lacking these proteins are specifically defective in the accumulation of subcomplex A. A total of 10 mutants lacking subcomplex A, including crr27/cpn60ß4, which is specifically defective in the folding of NdhH, and four mutants lacking NdhL-NdhO subunits, were extensively characterized. We propose a model for subcomplex A assembly: CRR41, NdhO, and native NdhH, as well as unknown factors, are first assembled to form an NDH subcomplex A assembly intermediate (NAI500). Subsequently, NdhJ, NdhM, NdhK, and NdhI are incorporated into NAI500 to form NAI400. CRR1, CRR6, and CRR42 are involved in this process. CRR7 is likely to be involved in the final step, in which the fully assembled NAI, including NdhN, is inserted into thylakoids.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cell Nucleus/genetics , Chloroplasts/metabolism , NADH Dehydrogenase/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Chloroplasts/enzymology , Molecular Sequence Data , NADH Dehydrogenase/genetics , Protein Binding , Tandem Mass SpectrometryABSTRACT
To gain insights into the molecular details of photosystem I (PSI) biogenesis, we characterized the PsbP-domain protein1 (ppd1) mutant of Arabidopsis thaliana that specifically lacks PSI activity. Deletion of PPD1 results in an inability of the mutant to grow photoautotrophically and a specific loss of the stable PSI complex. Unaltered transcription and translation of plastid-encoded PSI genes indicate that PPD1 acts at the posttranslational level. In vivo protein labeling experiments reveal that the rate of synthesis of PSI reaction center proteins PsaA/B in ppd1 is comparable to that of wild-type plants, whereas the rate of turnover of PsaA/B proteins is higher in ppd1 than in wild-type plants. With increasing leaf age, PPD1 content decreases considerably, while PSI content remains constant. PPD1 is a nuclear-encoded thylakoid lumenal protein and is associated with PSI but is not an integral subunit of PSI. Biochemical and molecular analyses reveal that PPD1 interacts directly and specifically with PsaB and PsaA. Yeast two-hybrid experiments show that PPD1 interacts with some lumenal loops of PsaB and PsaA. Our results suggest that PPD1 is a PSI assembly factor that assists the proper folding and integration of PsaB and PsaA into the thylakoid membrane.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Photosystem I Protein Complex/metabolism , Thylakoids/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Photosystem I Protein Complex/genetics , Thylakoids/geneticsABSTRACT
Some subunits of chloroplast NAD(P)H dehydrogenase (NDH) are related to those of the respiratory complex I, and NDH mediates photosystem I (PSI) cyclic electron flow. Despite extensive surveys, the electron donor and its binding subunits have not been identified. Here, we identified three novel components required for NDH activity. CRRJ and CRRL are J- and J-like proteins, respectively, and are components of NDH subcomplex A. CRR31 is an Src homology 3 domain-like fold protein, and its C-terminal region may form a tertiary structure similar to that of PsaE, a ferredoxin (Fd) binding subunit of PSI, although the sequences are not conserved between CRR31 and PsaE. Although CRR31 can accumulate in thylakoids independently of NDH, its accumulation requires CRRJ, and CRRL accumulation depends on CRRJ and NDH. CRR31 was essential for the efficient operation of Fd-dependent plastoquinone reduction in vitro. The phenotype of crr31 pgr5 suggested that CRR31 is required for NDH activity in vivo. We propose that NDH functions as a PGR5-PGRL1 complex-independent Fd:plastoquinone oxidoreductase in chloroplasts and rename it the NADH dehydrogenase-like complex.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Chloroplasts/enzymology , Ferredoxins/metabolism , NADH Dehydrogenase/metabolism , src Homology Domains , Arabidopsis Proteins/genetics , Binding Sites , Conserved Sequence/genetics , Intracellular Membranes/metabolism , Multienzyme Complexes , Mutation/genetics , NADH Dehydrogenase/chemistry , Oxidation-Reduction , Phototrophic Processes , Plastoquinone/metabolism , Structure-Activity Relationship , Thylakoids/metabolismABSTRACT
Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60ß4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60ß1-ß3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60ß subunits cannot complement the function of Cpn60ß4. Furthermore, the unique C-terminus of Cpn60ß4 is required for the full activity of the unique Cpn60 complex containing Cpn60ß4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates.
Subject(s)
Arabidopsis/metabolism , Chaperonin 60/metabolism , Chloroplasts/metabolism , Protein Folding , Amino Acid Sequence , Chaperonin 60/chemistry , Chaperonin 60/genetics , Chromatography, Affinity , Gene Expression Regulation, Plant , Molecular Sequence Data , Mutation , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.
Subject(s)
Photosystem II Protein Complex , Thylakoids , Chloroplasts/metabolism , Photosystem II Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Ribosomes/metabolism , Thylakoids/metabolismABSTRACT
Cyanobacterial NdhM, an oxygenic photosynthesis-specific NDH-1 subunit, has been found to be essential for the formation of a large complex of NDH-1 (NDH-1L). The cryo-electron microscopic (cryo-EM) structure of NdhM from Thermosynechococcus elongatus showed that the N-terminus of NdhM contains three ß-sheets, while two α-helixes are present in the middle and C-terminal part of NdhM. Here, we obtained a mutant of the unicellular cyanobacterium Synechocystis 6803 expressing a C-terminal truncated NdhM subunit designated NdhMΔC. Accumulation and activity of NDH-1 were not affected in NdhMΔC under normal growth conditions. However, the NDH-1 complex with truncated NdhM is unstable under stress. Immunoblot analyses showed that the assembly process of the cyanobacterial NDH-1L hydrophilic arm was not affected in the NdhMΔC mutant even under high temperature. Thus, our results indicate that NdhM can bind to the NDH-1 complex without its C-terminal α-helix, but the interaction is weakened. NDH-1L with truncated NdhM is more prone to dissociation, and this is particularly evident under stress conditions.