Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.     

Isolation and compositional analysis of a CP12-associated complex of calvin cycle enzymes from Nicotiana tabacum

Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a multiprotein complex with CP12, a small intrinsically-unstructured protein. Under oxidizing conditions, association with CP12 confers redox-sensitivity to the otherwise redox-insensitive A isoform of GAPDH (GapA) and provides an additional level of down-regulation to the redox-regulated PRK. To determine if CP12-mediated regulation is specific for GAPDH and PRK in vivo, a high molecular weight complex containing CP12 was isolated from tobacco chloroplasts and leaves and its protein composition was characterized. Gel electrophoresis and immunoblot analyses after separation of stromal proteins by size fractionation verified that the GAPDH (both isoforms) and PRK co-migrated with CP12 in dark- but not light-adapted chloroplasts. Nano-liquid-chromatography-mass-spectrometry of the isolated complex identified only CP12, GAPDH and PRK. Since nearly all of the CP12 from darkened chloroplasts migrates with GADPH and PRK as a high molecular mass species, these data indicate that the tight association of tobacco CP12 with GAPDH and PRK is specific and involves no other Calvin Cycle enzymes.     

Reconstitution and properties of the recombinant glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase supramolecular complex of Arabidopsis

Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the regulatory peptide CP12 a supramolecular complex in Arabidopsis (Arabidopsis thaliana) that could be reconstituted in vitro using purified recombinant proteins. Both enzyme activities were strongly influenced by complex formation, providing an effective means for regulation of the Calvin cycle in vivo. PRK and CP12, but not GapA (A(4) isoform of GAPDH), are redox-sensitive proteins. PRK was reversibly inhibited by oxidation. CP12 has no enzymatic activity, but it changed conformation depending on redox conditions. GapA, a bispecific NAD(P)-dependent dehydrogenase, specifically formed a binary complex with oxidized CP12 when bound to NAD. PRK did not interact with either GapA or CP12 singly, but oxidized PRK could form with GapA/CP12 a stable ternary complex of about 640 kD (GapA/CP12/PRK). Exchanging NADP for NAD, reducing CP12, or reducing PRK were all conditions that prevented formation of the complex. Although GapA activity was little affected by CP12 alone, the NADPH-dependent activity of GapA embedded in the GapA/CP12/PRK complex was 80% inhibited in respect to the free enzyme. The NADH activity was unaffected. Upon binding to GapA/CP12, the activity of oxidized PRK dropped from 25% down to 2% the activity of the free reduced enzyme. The supramolecular complex was dissociated by reduced thioredoxins, NADP, 1,3-bisphosphoglycerate (BPGA), or ATP. The activity of GapA was only partially recovered after complex dissociation by thioredoxins, NADP, or ATP, and full GapA activation required BPGA. NADP, ATP, or BPGA partially activated PRK, but full recovery of PRK activity required thioredoxins. The reversible formation of the GapA/CP12/PRK supramolecular complex provides novel possibilities to finely regulate GapA ("non-regulatory" GAPDH isozyme) and PRK (thioredoxin sensitive) in a coordinated manner.     

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.     

The Calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions

In Synechococcus PCC7942 cells grown in the dark, the concentrations of NAD(H) and NADP(H) were 128+/-2.5 and 483+/-4.0 microm, respectively, while those in the cells under light conditions were 100+/-5.0 and 649+/-7.0 microm, respectively. Analysis of gel filtration indicated that the change of the ratio of NADP(H) to NAD(H) in cyanobacterial cells under light/dark conditions controls the reversible dissociation of the PRK/CP12/GAPDH complex (approximately 520 kDa) consisting of phosphoribulokinase (PRK), CP12, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). S. 7942 CP12 lacked the two Cys residues essential for formation of the N-terminal peptide loop in the CP12 of higher plants, but the N-terminal region of S. 7942 CP12 had the ability to be associated with PRK. The growth of mutant cells in which the CP12 gene was disrupted by a kanamycin resistance cartridge gene was almost the same as that of wild-type cells under continuous light conditions. However, under the light/dark cycle (12 h/12 h), the growth of CP12-disrupted mutant cells was significantly inhibited compared with that of wild-type cells. The mutant cells showed a decreased rate of O2 consumption and an increased level of ribulose 1,5-bisphosphate compared with wild-type cells in the dark. These data suggest that under light and dark conditions, the oligomerization of CP12 with PRK and GAPDH regulates the activities of both enzymes and thus the carbon flow from the Calvin cycle to the oxidative pentose phosphate cycle.     

The small protein CP12: a protein linker for supramolecular complex assembly

CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants. It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12. The role of CP12 in this complex assembly has not been determined. To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli. Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12. On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). This unit probably dimerizes due partially to interactions between the enzymes of each unit. Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions. Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured. Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly. CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants.     

Modulation,via protein-protein interactions,of glyceraldehyde-3-phosphate dehydrogenase activity through redox phosphoribulokinase regulation

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) embedded in the phosphoribulokinase (PRK).GAPDH.CP12 complex was increased 2-3-fold by reducing agents. This occurred by interaction with PRK as the cysteinyl sulfhydryls (4 SH/subunit) of GAPDH within the complex were unchanged whatever the redox state of the complex. But isolated GAPDH was not activated. Alkylation plus mass spectrometry also showed that PRK had one disulfide bridge and three SH groups per monomer in the active oxidized complex. Reduction disrupted this disulfide bridge to give 2 more SH groups and a much more active enzyme. We assessed the kinetics and dynamics of the interactions between PRK and GAPDH/CP12 using biosensors to measure complex formation in real time. The apparent equilibrium binding constant for GAPDH/CP12 and PRK was 14 +/- 1.6 nm for oxidized PRK and 62 +/- 10 nm for reduced PRK. These interactions were neither pH- nor temperature-dependent. Thus, the dynamics of PRK.GAPDH.CP12 complex formation and GAPDH activity are modulated by the redox state of PRK.     

Structure basis for the regulation of glyceraldehyde-3-phosphate dehydrogenase activity via the intrinsically disordered protein CP12

The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.     

Spontaneous assembly of photosynthetic supramolecular complexes as mediated by the intrinsically unstructured protein CP12

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-GAPDH)-(CP12-2)(2) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-GAPDH)-(CP12-2)(2)-(PRK))(2). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAPDH and PRK during darkness in photosynthetic tissues.     

Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation.

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.     

Conformational selection and folding-upon-binding of intrinsically disordered protein CP12 regulate photosynthetic enzymes assembly

Carbon assimilation in plants is regulated by the reduction of specific protein disulfides by light and their re-oxidation in the dark. The redox switch CP12 is an intrinsically disordered protein that can form two disulfide bridges. In the dark oxidized CP12 forms an inactive supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase, two enzymes of the carbon assimilation cycle. Here we show that binding of CP12 to GAPDH, the first step of ternary complex formation, follows an integrated mechanism that combines conformational selection with induced folding steps. Initially, a CP12 conformation characterized by a circular structural motif including the C-terminal disulfide is selected by GAPDH. Subsequently, the induced folding of the flexible C-terminal tail of CP12 in the active site of GAPDH stabilizes the binary complex. Formation of several hydrogen bonds compensates the entropic cost of CP12 fixation and terminates the interaction mechanism that contributes to carbon assimilation control.     

Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts.

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP- GAPDH-A2B2) involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP-GAPDH and PRK and (b) of only NADP-GAPDH, respectively, in darkened chloroplasts. While the 550-kDa PRK/GAPDH/CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation with dithiothreitol and the effector 1,3-bisphosphoglycerate. In the light, PRK is therefore completely in its activated state under all conditions, even in low light, while GAPDH activation in the light is characterized by a two-step mechanism with 60-70% activation under most conditions in the light, and the activation of the remaining 30-40% occurring only when 1,3-bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP-GAPDH, some of the PRK reaggregates. Recombinant GapA/GapB constructs form the A8B8 complex immediately upon expression in E. coli.     

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Regulation of the Calvin–Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin–Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A₂B₂-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A₄-GAPDH. GapB subunits of A₂B₂-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A₂B₂-GAPDH into strongly inactivated A₈B₈-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A₄-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A₂B₂-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.     

Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH/CP12 complex. Consequently, a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central alpha-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH/CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5-75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcal.mol(-1) destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.     

Comparative sequence analysis of CP12, a small protein involved in the formation of a Calvin cycle complex in photosynthetic organisms

CP12, a small intrinsically unstructured protein, plays an important role in the regulation of the Calvin cycle by forming a complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An extensive search in databases revealed 129 protein sequences from, higher plants, mosses and liverworts, different groups of eukaryotic algae and cyanobacteria. CP12 was identified throughout the Plantae, apart from in the Prasinophyceae. Within the Chromalveolata, two putative CP12 proteins have been found in the genomes of the diatom Thalassiosira pseudonana and the haptophyte Emiliania huxleyi, but specific searches in further chromalveolate genomes or EST datasets did not reveal any CP12 sequences in other Prymnesiophyceae, Dinophyceae or Pelagophyceae. A species from the Euglenophyceae within the Excavata also appeared to lack CP12. Phylogenetic analysis showed a clear separation into a number of higher taxonomic clades and among different forms of CP12 in higher plants. Cyanobacteria, Chlorophyceae, Rhodophyta and Glaucophyceae, Bryophyta, and the CP12-3 forms in higher plants all form separate clades. The degree of disorder of CP12 was higher in higher plants than in the eukaryotic algae and cyanobacteria apart from the green algal class Mesostigmatophyceae, which is ancestral to the streptophytes. This suggests that CP12 has evolved to become more flexible and possibly take on more general roles. Different features of the CP12 sequences in the different taxonomic groups and their potential functions and interactions in the Calvin cycle are discussed.     

Regulation of phosphoribulokinase and glyceraldehyde 3‐phosphate dehydrogenase in a freshwater diatom,Asterionella formosa1

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.     

CP12: a small nuclear-encoded chloroplast protein provides novel insights into higher-plant GAPDH evolution

Higher-plant chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) is composed of two different nuclear-encoded subunits, GAPA and GAPB, forming the highly active heterotetrameric A₂B₂ enzyme. The main difference between these two subunits is a C-terminal extension of about 30 amino acid residues of GAPB. We present cDNA clones for a nuclear-encoded chloroplast protein from pea, spinach and tobacco, which we have named CP12. The mature protein consists of only 74, 75 and 76 amino acid residues, respectively and contains two domains with significant homology to the C-terminal extension of GAPB. Affinity chromatography approaches reveal also a specific interaction between CP12 and chloroplast GAPDH. Northern blot analysis indicates that CP12 is, like plastid GAPDH, expressed in green and also in etiolated leaves. Further homology is observed between CP12 and ORF3, an open reading frame located in the hox gene cluster of Anabaena variabilis. This gene cluster encodes the subunits of the bidirectional NADP⁺-dependent [NiFeS] dehydrogenase. We propose therefore a common evolutionary origin of CP12 and higher-plant chloroplast GAPDH subunit GAPB from the cyanobacterial ORF3.