Chloroplast ATP synthase biogenesis requires peripheral stalk subunits AtpF and ATPG and stabilization of atpE mRNA by OPR protein MDE1.

Chloroplast ATP synthase contains subunits of plastid and nuclear genetic origin. To investigate the coordinated biogenesis of this complex, we isolated novel ATP synthase mutants in the green alga Chlamydomonas reinhardtii by screening for high light sensitivity. We report here the characterization of mutants affecting the two peripheral stalk subunits b and b', encoded respectively by the atpF and ATPG genes, and of three independent mutants which identify the nuclear factor MDE1, required to stabilize the chloroplast-encoded atpE mRNA. Whole-genome sequencing revealed a transposon insertion in the 3'UTR of ATPG while mass spectrometry shows a small accumulation of functional ATP synthase in this knock-down ATPG mutant. In contrast, knock-out ATPG mutants, obtained by CRISPR-Cas9 gene editing, fully prevent ATP synthase function and accumulation, as also observed in an atpF frame-shift mutant. Crossing ATP synthase mutants with the ftsh1-1 mutant of the major thylakoid protease identifies AtpH as an FTSH substrate, and shows that FTSH significantly contributes to the concerted accumulation of ATP synthase subunits. In mde1 mutants, the absence of atpE transcript fully prevents ATP synthase biogenesis and photosynthesis. Using chimeric atpE genes to rescue atpE transcript accumulation, we demonstrate that MDE1, a novel octotricopeptide repeat (OPR) protein, genetically targets the atpE 5'UTR. In the perspective of the primary endosymbiosis (~1.5 Gy), the recruitment of MDE1 to its atpE target exemplifies a nucleus/chloroplast interplay that evolved rather recently, in the ancestor of the CS clade of Chlorophyceae, ~300 My ago.

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit ß.

Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit ß in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPß may be involved in proper ATP synthase accumulation during complex assembly.

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.

Enhanced abundance and activity of the chloroplast ATP synthase in rice through the overexpression of the AtpD subunit.

ATP, produced by the light reactions of photosynthesis, acts as the universal cellular energy cofactor fuelling all life processes. Chloroplast ATP synthase produces ATP using the proton motive force created by solar energy-driven thylakoid electron transport reactions. Here we investigate how increasing abundance of ATP synthase affects leaf photosynthesis and growth of rice, Oryza sativa variety Kitaake. We show that overexpression of AtpD, the nuclear-encoded subunit of the chloroplast ATP synthase, stimulates both abundance of the complex, confirmed by immunodetection of thylakoid complexes separated by Blue Native-PAGE, and ATP synthase activity, detected as higher proton conductivity of the thylakoid membrane. Plants with increased AtpD content had higher CO2 assimilation rates when a stepwise increase in CO2 partial pressure was imposed on leaves at high irradiance. Fitting of the CO2 response curves of assimilation revealed that plants overexpressing AtpD had a higher electron transport rate (J) at high CO2, despite having wild-type-like abundance of the cytochrome b6f complex. A higher maximum carboxylation rate (Vcmax) and lower cyclic electron flow detected in transgenic plants both pointed to an increased ATP production compared with wild-type plants. Our results present evidence that the activity of ATP synthase modulates the rate of electron transport at high CO2 and high irradiance.

Mitochondrial movement during its association with chloroplasts in Arabidopsis thaliana.

Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis.

Positive Selection in the Chloroplastic ATP-Synthase ß-Subunit and Its Relation to Virulence Factors.

The most paradigmatic examples of molecular evolution under positive selection involve genes related to the immune system. Recently, different chloroplastic factors have been shown to be important for plant defenses, among them, the α- and ß-subunits of the ATP synthase. The ß-subunit has been reported to interact with several viral proteins while both proteins have been implicated with sensitivity to tentoxin, a phytotoxin produced by the widespread fungus Alternaria alternata. Given the relation of both protein to virulence factors, we studied whether these proteins are evolving under positive selection. To this end, we used the dN/dS ratio to examine possible sites under positive selection in several Angiosperm clades. After examining 79 plant genera and 1232 species, we found three times more sites under pervasive diversifying selection in the N-terminal region of the ß-subunit compared to the α-subunit, supporting previous results which identified this region as responsible for interacting with viral proteins. Moreover, we found the site 83 of ß-subunit under positive selection in several plant genera, a site clearly related to the sensitivity to tentoxin according to biochemistry assays, which possibly reflects the selective pressure of the non-host specific tentoxin across various Angiosperm clades.

H+ Transport by K+ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

The OPR Protein MTHI1 Controls the Expression of Two Different Subunits of ATP Synthase CFo in Chlamydomonas reinhardtii.

In the green alga Chlamydomonas (Chlamydomonas r einhardtii), chloroplast gene expression is tightly regulated posttranscriptionally by gene-specific trans-acting protein factors. Here, we report the identification of the octotricopeptide repeat protein MTHI1, which is critical for the biogenesis of chloroplast ATP synthase oligomycin-sensitive chloroplast coupling factor. Unlike most trans-acting factors characterized so far in Chlamydomonas, which control the expression of a single gene, MTHI1 targets two distinct transcripts: it is required for the accumulation and translation of atpH mRNA, encoding a subunit of the selective proton channel, but it also enhances the translation of atpI mRNA, which encodes the other subunit of the channel. MTHI1 targets the 5' untranslated regions of both the atpH and atpI genes. Coimmunoprecipitation and small RNA sequencing revealed that MTHI1 binds specifically a sequence highly conserved among Chlorophyceae and the Ulvale clade of Ulvophyceae at the 5' end of triphosphorylated atpH mRNA. A very similar sequence, located ∼60 nucleotides upstream of the atpI initiation codon, was also found in some Chlorophyceae and Ulvale algae species and is essential for atpI mRNA translation in Chlamydomonas. Such a dual-targeted trans-acting factor provides a means to coregulate the expression of the two proton hemi-channels.

MDA1, a nucleus-encoded factor involved in the stabilization and processing of the atpA transcript in the chloroplast of Chlamydomonas.

In Chlamydomonas reinhardtii, chloroplast gene expression is tightly regulated post-transcriptionally by gene-specific trans-acting protein factors. Here, we report the molecular identification of an OctotricoPeptide Repeat (OPR) protein, MDA1, which governs the maturation and accumulation of the atpA transcript, encoding subunit α of the chloroplast ATP synthase. As does TDA1, another OPR protein required for the translation of the atpA mRNA, MDA1 targets the atpA 5'-untranslated region (UTR). Unexpectedly, it binds within a region of approximately 100 nt in the middle of the atpA 5'-UTR, at variance with the stabilization factors characterized so far, which bind to the 5'-end of their target mRNA to protect it from 5' → 3' exonucleases. It binds the same region as TDA1, with which it forms a high-molecular-weight complex that also comprises the atpA mRNA. This complex dissociates upon translation, promoting degradation of the atpA mRNA. We suggest that atpA transcripts, once translated, enter the degradation pathway because they cannot reassemble with MDA1 and TDA1, which preferentially bind to de novo transcribed mRNAs.

Nucleus-Encoded Protein BFA1 Promotes Efficient Assembly of the Chloroplast ATP Synthase Coupling Factor 1.

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.

Effects of rare earth and acid rain pollution on plant chloroplast ATP synthase and element contents at different growth stages.

Combined rare earth and acid rain pollution has become a new environmental problem, seriously affecting plant survival. The effects of these two kinds of pollutants on plant photosynthesis have been reported, but the micro mechanisms are not very clear. In this research, we studied the effects of lanthanum [La(III), 0.08, 1.20 and 2.40 mM] and acid rain (pH value = 2.5, 3.5 and 4.5) on the ATPase activity and gene transcription level and the functional element contents in rice leaf chloroplasts. The results showed that the combined 0.08 mM La(III) and pH 4.5 acid rain increased the ATPase activity and gene transcription level as well as contents of some functional elements. But other combined treatments of acid rain and La(III) reduced the ATPase activity and gene transcription level as well as functional element contents. The change magnitude of the above indexes at rice booting stage was greater than that in seedling stage or grain filling stage. These results reveal that effects of La(III) and acid rain on ATPase activity and functional element contents in rice leaf chloroplasts are related to the combination of La(III) dose and acid rain intensity and the plant growth stage. In addition, the changes in the ATPase activity were related to ATPase gene transcription level. This study would provide a reference for understanding the microcosmic mechanism of rare earth and acid rain pollution on plant photosynthesis and contribute to evaluate the possible environmental risks associated with combined La(III) and acid rain pollution. ONE SENTENCE SUMMARY: The effects of La(III) and acid rain on activity and gene transcription level of rice chloroplast ATPase and contents of functional elements were different at different growth stages.

A γ-subunit point mutation in Chlamydomonas reinhardtii chloroplast F1Fo-ATP synthase confers tolerance to reactive oxygen species.

The chloroplast F1Fo-ATP synthase (CF1Fo) drives ATP synthesis and the reverse reaction of ATP hydrolysis. The enzyme evolved in a cellular environment where electron transfer processes and molecular oxygen are abundant, and thiol modulation in the γ-subunit via thioredoxin is important for its ATPase activity regulation. Especially under high light, oxygen can be reduced and forms reactive oxygen species (ROS) which can oxidize CF1Fo among various other biomolecules. Mutation of the conserved ROS targets resulted in a tolerant enzyme, suggesting that ROS might play a regulatory role. The mutations had several side effects in vitro, including disturbance of the ATPase redox regulation [F. Buchert et al., Biochim. Biophys. Acta, 1817 (2012) 2038-2048]. This would prevent disentanglement of thiol- and ROS-specific modes of regulation. Here, we used the F1 catalytic core in vitro to identify a point mutant with a functional ATPase redox regulation and increased H2O2 tolerance. In the next step, the mutation was introduced into Chlamydomonas reinhardtii CF1Fo, thereby allowing us to study the physiological role of ROS regulation of the enzyme in vivo. We demonstrated in high light experiments that CF1Fo ROS targets were involved in the significant inhibition of ATP synthesis rates. Molecular events upon modification of CF1Fo by ROS will be considered.

Chloroplastic ATP synthase builds up a proton motive force preventing production of reactive oxygen species in photosystem I.

Over-reduction of the photosynthetic electron transport (PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species (ROS) within photosystem I (PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H+ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over-reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate (EMS)-treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over-reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ-subunit of chloroplastic CF0 CF1 -ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H+ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF0 CF1 -ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild-type (WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF0 CF1 -ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H+ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF0 CF1 -ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis.

A Nucleus-Encoded Chloroplast Protein YL1 Is Involved in Chloroplast Development and Efficient Biogenesis of Chloroplast ATP Synthase in Rice.

Chloroplast ATP synthase (cpATPase) is an importance thylakoid membrane-associated photosynthetic complex involved in the light-dependent reactions of photosynthesis. In this study, we isolated and characterized a rice (Oryza sativa) mutant yellow leaf 1 (yl1), which exhibits chlorotic leaves throughout developmental stages. The YL1 mutation showed reduced chlorophyll contents, abnormal chloroplast morphology, and decreased photochemical efficiency. Moreover, YL1 deficiency disrupts the expression of genes associated with chloroplast development and photosynthesis. Molecular and genetic analyses revealed that YL1 is a nucleus-encoded protein with a predicted transmembrane domain in its carboxyl-terminus that is conserved in the higher plant kingdom. YL1 localizes to chloroplasts and is preferentially expressed in green tissues containing chloroplasts. Immunoblot analyses showed that inactivation of YL1 leads to drastically reduced accumulation of AtpA (α) and AtpB (ß), two core subunits of CF1αß subcomplex of cpATPase, meanwhile, a severe decrease (ca. 41.7%) in cpATPase activity was observed in the yl1-1 mutant compared with the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays revealed a specific interaction between YL1 and AtpB subunit of cpATPase. Taken together, our results suggest that YL1 is a plant lineage-specific auxiliary factor involved in the biogenesis of the cpATPase complex, possibly via interacting with the ß-subunit.

The Arabidopsis Protein CGLD11 Is Required for Chloroplast ATP Synthase Accumulation.

ATP synthases in chloroplasts (cpATPase) and mitochondria (mtATPase) are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multisubunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 (CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11) is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLD11 function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the ß subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential.

Expression of ATP synthase CF1 alpha subunit gene (CTL-spn) as screened by the cDNA-SRAP approach is correlated with spininess in Carthamus tinctorius L.

The safflower floret is a traditional Chinese medicine used to promote blood circulation and remove obstruction in the channels. The spines on its bracts are considered a handicap when manual harvest is involved. In this study, cDNA-SRAP was used to systematically investigate which genes are associated with the spines. Sixty pairs of possible primer combinations were used on two cDNA pools representing spininess and spinelessness. Six transcript-derived fragments were identified, of which two with low recombination were sequenced successfully and named as GPY-1 and GPY-2. By using the RACE method, the full-length cDNA of GPY-2 is cloned and named as CTL-spn. The full-length cDNA of CTL-spn was 1 679 bp long with a 1 524 bp ORF encoding a 508 aminoacid protein. The deduced amino acid sequence of the CTL-spn gene shared a high homology (97%) with other known ATP synthase CF1 alpha subunits. Semiquantitative RT-PCR analysis revealed that the mRNA of GPY-1 and GPY-2 accumulated in only spiny lines. Considering the important role of ATP synthase CF1 alpha subunit in plants, it may directly take part in the formation process of spininess and enhancing resistance reaction of spiny safflower. Also, our results provide the important insights for breeding spineless cultivars of safflower.

Interaction between PVY HC-Pro and the NtCF1ß-subunit reduces the amount of chloroplast ATP synthase in virus-infected tobacco.

The photosynthetic rate of virus-infected plants is always reduced. However, the molecular mechanism underlying this phenomenon remains unclear. The helper component-proteinase (HC-Pro) of Potato virus Y (PVY) was found in the chloroplasts of PVY-infected tobacco, indicating some new function of HC-Pro in the chloroplasts. We generated HC-Pro transgenic plants with a transit peptide to target the protein to chloroplast. The HC-Pro transgenic tobacco showed a decreased photosynthetic rate by 25% at the light intensity of 600 µmol m(-2) s(-1). Using a yeast two-hybrid screening assay to search for chloroplast proteins interacting with HC-Pro, we identified that PVY HC-Pro can interact with the chloroplast ATP synthase NtCF1ß-subunit. This interaction was confirmed by GST pull-down and co-immunoprecipitation assays. HC-Pro didn't interfere with the activity of assembled ATP synthase in vitro. The HC-Pro/NtCF1ß-subunit interaction might affect the assembly of ATP synthase complex. Quantitative western blot and immunogold labeling of the ATP synthase indicated that the amount of ATP synthase complex was decreased in both the HC-Pro transgenic and the PVY-infected tobacco. These results demonstrate that HC-Pro plays an important role in reducing the photosynthetic rate of PVY-infected plants, which is a completely new role of HC-Pro besides its multiple known functions.

The thylakoid membrane protein CGL160 supports CF1CF0 ATP synthase accumulation in Arabidopsis thaliana.

The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF1CF0 complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF1 sub-complex. We suggest therefore that it may facilitate the assembly of CF1 into the holocomplex.

PAB is an assembly chaperone that functions downstream of chaperonin 60 in the assembly of chloroplast ATP synthase coupling factor 1.

The chloroplast ATP synthase, a multisubunit complex in the thylakoid membrane, catalyzes the light-driven synthesis of ATP, thereby supplying the energy for carbon fixation during photosynthesis. The chloroplast ATP synthase is composed of both nucleus- and chloroplast-encoded proteins that have required the evolution of novel mechanisms to coordinate the biosynthesis and assembly of chloroplast ATP synthase subunits temporally and spatially. Here we have elucidated the assembly mechanism of the α3ß3γ core complex of the chloroplast ATP synthase by identification and functional characterization of a key assembly factor, PAB (protein in chloroplast atpase biogenesis). PAB directly interacts with the nucleus-encoded γ subunit and functions downstream of chaperonin 60 (Cpn60)-mediated CF1γ subunit folding to promote its assembly into the catalytic core. PAB does not have any recognizable motifs or domains but is conserved in photosynthetic eukaryotes. It is likely that PAB evolved together with the transfer of chloroplast genes into the nucleus to assist nucleus-encoded CF1γ assembly into the CF1 core. Such coordination might represent an evolutionarily conserved mechanism for folding and assembly of nucleus-encoded proteins to ensure proper assembly of multiprotein photosynthetic complexes.