Glyceraldehyde-3-phosphate Dehydrogenase Isoform Research Articles

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Characterization of LcGAPC and its transcriptional response to salt and alkali stress in two ecotypes of Leymus chinensis (Trin.) Tzvelev

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a housekeeping protein which plays various roles in non-metabolic processes in addition to its role in glucose catabolism. There are several GAPDH isoforms in plant cells. In this study, we cloned the full-length cDNA encoding the isoform GAPC in two ecotypes of Leymus chinensis (Trin.) Tzvelev, gray–green type and yellow–green type. The LcGAPC sequence includes an open reading frame (ORF) of 1014 bp encoding 337 amino acids. The predicted molecular weight is 36.5 kDa, and the pI is 6.19. The mRNA expression of LcGAPC decreased at 24 h and then increased significantly after 7 d after 400 mmol/L NaCl stress. At 200 mmol/L mixed alkali stress (NaHCO3:Na2CO3=9:1), the LcGAPC expression level gradually increased as time increased in the two ecotypes of L. chinensis. The results suggested that LcGAPC is a stress-inducible gene that might play a role in the salt and alkali stress response. This study provided a basis to further study the mechanism of expression characteristics under salt and alkali stress conditions of L. chinensis.

The intriguing CP12-like tail of adenylate kinase 3 from Chlamydomonas reinhardtii.

Adenylate kinases (ADK) are key enzymes that maintain the energetic balance in cellular compartments by catalyzing the reaction: AMP + ATP↔2 ADP. Here, we analyzed the chloroplast ADK 3 from the green alga, Chlamydomonas reinhardtii for the first time. This enzyme bears a C-terminal extension that is highly similar to the C-terminal end of the intrinsically disordered protein CP12 that plays a major role in the redox regulation of key enzymes of the Calvin-Benson cycle like glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase. The only other known example of a CP12-like extension is found in the GapB isoform of GAPDH, where it is responsible for the autonomous redox regulation of the higher plant A2 B2 GAPDH. In this study, we show that the CP12-like tail is not involved in the redox regulation of ADK 3, but contributes greatly to its stability, and is essential for the post-translational modification of the Cys221 residue by glutathione. This report highlights the fact that the C-terminal part of the CP12 protein can act as a moonlighting, intrinsically disordered module conferring additional capabilities to the proteins to which it is added. Adenylate kinase (ADK, EC 2.7.4.3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13).

Beyond glycolysis: GAPDHs are multi-functional enzymes involved in regulation of ROS, autophagy, and plant immune responses.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in energy metabolism with diverse cellular regulatory roles in vertebrates, but few reports have investigated the importance of plant GAPDH isoforms outside of their role in glycolysis. While animals possess one GAPDH isoform, plants possess multiple isoforms. In this study, cell biological and genetic approaches were used to investigate the role of GAPDHs during plant immune responses. Individual Arabidopsis GAPDH knockouts (KO lines) exhibited enhanced disease resistance phenotypes upon inoculation with the bacterial plant pathogen Pseudomonas syringae pv. tomato. KO lines exhibited accelerated programmed cell death and increased electrolyte leakage in response to effector triggered immunity. Furthermore, KO lines displayed increased basal ROS accumulation as visualized using the fluorescent probe H2DCFDA. The gapa1-2 and gapc1 KOs exhibited constitutive autophagy phenotypes in the absence of nutrient starvation. Due to the high sequence conservation between vertebrate and plant cytosolic GAPDH, our experiments focused on cytosolic GAPC1 cellular dynamics using a complemented GAPC1-GFP line. Confocal imaging coupled with an endocytic membrane marker (FM4-64) and endosomal trafficking inhibitors (BFA, Wortmannin) demonstrated cytosolic GAPC1 is localized to the plasma membrane and the endomembrane system, in addition to the cytosol and nucleus. After perception of bacterial flagellin, GAPC1 dynamically responded with a significant increase in size of fluorescent puncta and enhanced nuclear accumulation. Taken together, these results indicate that plant GAPDHs can affect multiple aspects of plant immunity in diverse sub-cellular compartments.

Transglutaminase 2-dependent Deamidation of Glyceraldehyde-3-phosphate Dehydrogenase Promotes Trophoblastic Cell Fusion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.

Moonlighting cell-surface GAPDH recruits apotransferrin to effect iron egress from mammalian cells.

Iron (Fe(2+), Fe(3+)) homeostasis is a tightly regulated process, involving precise control of iron influx and egress from cells. Although the mechanisms of its import into cells by iron carrier molecules are well characterized, iron export remains poorly understood. The current paradigm envisages unique functions associated with specialized macromolecules for its cellular import (transferrin receptors) or export (ferroportin, also known as SLC40A1). Previous studies have revealed that iron-depleted cells recruit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multitasking, 'moonlighting' protein, to their surface for internalization of the iron carrier holotransferrin. Here, we report that under the converse condition of intracellular iron excess, cells switch the isoform of GAPDH on their surface to one that now recruits iron-free apotransferrin in close association with ferroportin to facilitate the efflux of iron. Increased expression of surface GAPDH correlated with increased apotransferrin binding and enhanced iron export from cells, a capability lost in GAPDH-knockdown cells. These findings were confirmed in vivo utilizing a rodent model of iron overload. Besides identifying for the first time an apotransferrin receptor, our work uncovers the two-way switching of multifunctional molecules to manage cellular micronutrient requirements.

Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional) properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively modified GAPDH in stress signaling pathways in plants.

Proteomic evaluation of myofibrillar carbonylation in chilled fish mince and its inhibition by catechin

The present study investigates the susceptibility of individual myofibrillar proteins from mackerel (Scomber scombrus) mince to undergo carbonylation reactions during chilled storage, and the antioxidant capacity of (+)-catechin to prevent oxidative processes of proteins. The carbonylation of each particular protein was quantified by combining the labelling of protein carbonyls by fluorescein-5-thiosemicarbazide (FTSC) with 1-D or 2-D gel electrophoresis. Alpha skeletal actin, glycogen phosphorylase, unnamed protein product (UNP) similar to enolase, pyruvate kinase, isoforms of creatine kinase, aldolase A and an isoform of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed elevated oxidation in chilled non-supplemented mince. Myosin heavy chain (MHC) was not carbonylated in chilled muscle, but an extensive MHC degradation was observed in those samples. The supplementation of catechin reduced protein oxidation and lipid oxidation in a concentration-dependent manner: control>25>100≈200ppm. Therefore, the highest catechin concentrations (100 and 200ppm) exhibited the strongest antioxidant activity. Catechin (200ppm) reduced significantly carbonylation of protein spots identified as glycogen phosphorylase, pyruvate kinase muscle isozyme, isoforms of creatine kinase. Conversely, catechin was ineffective to inhibit the oxidation of actin and UNP similar to enolase. These results draw attention to the inefficiency of catechin to prevent actin oxidation, in contrast to the extremely high efficiency of catechin in inhibiting oxidation of lipids and other proteins.

Preliminary crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase 3 from Saccharomyces cerevisiae.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in the glycolytic pathway. In addition to its conventional metabolic role, GAPDH has been identified to possess diverse cellular functions. In this study, glyceraldehyde-3-phosphate dehydrogenase 3, the third isoform of GAPDH from Saccharomyces cerevisiae, was cloned, expressed, purified and crystallized. The crystals belonged to space group I4(1)22, with unit-cell parameters a=b=116.13, c = 119.21 Å. X-ray diffraction data were collected to a resolution of 2.6 Å. The structure was solved by molecular replacement and refinement is in progress.

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate <italic>Tetrahymena thermophila</italic>

In the present study, we purified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is involved in cellular energy production and has important housekeeping functions, from the ciliate Tetrahymena thermophila using a three-step procedure. The enzyme was purified ~68 folds by ammonium sulfate precipitation, followed by two steps of column chromatography (DEAE-cellulose and Mono-S). The purified enzyme is a homotetramer with a molecular weight of ~120 kDa. Isoelectric focusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blot analysis showed a single 32-kDa band corresponding to the enzyme subunit using a monospecific polyclonal antibody against the T. thermophila GAPDH. The maximum of enzyme activity occurred at pH 8.0 and at 30-35°C. The apparent K(m) values for both NAD(+) and D-glyceraldehyde-3-phosphate were 0.102 ± 0.012 and 0.360 ± 0.018 mM, respectively. The maximal velocity (V(max)) was 39.40 ± 2.95 U/mg. The T. thermophila GAPDH is inhibited by oxidative and nitrosative stress reagents.

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.

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.

Characterization of proteome alterations in Phanerochaete chrysosporium in response to lead exposure

BackgroundTotal soluble proteome alterations of white rot fungus Phanerochaete chrysosporium in response to different doses (25, 50 and 100 μM) of Pb (II) were characterized by 2DE in combination with MALDI-TOF-MS.ResultsDose-dependent molecular response to Pb (II) involved a total of 14 up-regulated and 21 down-regulated proteins. The induction of an isoform of glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase class V, mRNA splicing factor, ATP-dependent RNA helicase, thioredoxin reductase and actin required a Pb (II) dose of at least 50 μM. Analysis of the proteome dynamics of mid-exponential phase cells of P. chrysosporium subjected to 50 μM lead at exposure time intervals of 1, 2, 4 and 8 h, identified a total of 23 proteins in increased and 67 proteins in decreased amount. Overall, the newly induced/strongly up-regulated proteins involved in (i) amelioration of lipid peroxidation products, (ii) defense against oxidative damage and redox metabolism, (iii) transcription, recombination and DNA repair (iv) a yet unknown function represented by a putative protein.ConclusionThe present study implicated the particular role of the elements of DNA repair, post-tanscriptional regulation and heterotrimeric G protein signaling in response to Pb (II) stress as shown for the first time for a basidiomycete.

Differential modulations of two glyceraldehyde 3-phosphate dehydrogenase mRNAs in response to bacterial and viral challenges in a marine teleost Oplegnathus fasciatus (Perciformes)

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been recognized as an invariant internal control for various gene expression studies due to its classical housekeeping role in the glycolytic metabolism. However recently, this enzyme has been proven to be a multifunctional protein involved in diverse non-glycolytic activities. In the present study, two paralogue isoforms of GAPDH mRNAs were characterized from a marine teleost species, rockbream ( Oplegnathus fasciatus; Perciformes) and their transcriptional responses to bacterial and viral infections were examined. Rockbream GAPDH1 and GAPDH2 cDNAs encoded 333 and 335 amino acids, respectively, and the amino acid sequence identity between those two isoforms was 74%. Two isoform GAPDH mRNAs were detected ubiquitously in all of tissues examined, but their expression levels were quite variable among tissues. Based on the real-time RT-PCR analysis, the transcription of rbGAPDH1 was affected by neither bacterial ( Escherichia coli, Edwardsiella tarda, Vibrio anguillarum or Streptococcus iniae) nor viral (rockbream iridovirus; RBIV) challenges. However on the contrary, the mRNA expression of rbGAPDH2 was significantly up-regulated in liver resulting from the bacterial infections (up to 25-fold), and in both liver (more than sixfold) and kidney (up to fivefold) from the viral infection. Results in the present study suggest that teleost GAPDH isoforms may also be potentially involved in immune modulations especially with respect to inflammatory responses, which is distinct from its classical glycolytic function.

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.

Molecular mechanism of thioredoxin regulation in photosynthetic A 2 B 2 -glyceraldehyde-3-phosphate dehydrogenase

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A(4)-GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB-GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A(2)B(2)-GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A(2)B(2)-GAPDH and chimeric mutant [A+CTE](4)-GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A(2)B(2)-GAPDH and [A+CTE](4)-GAPDH, compared with fully active A(4)-GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB-GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo.

Purification and Characterization of Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase from the Dromedary Camel

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12), a key enzyme of carbon metabolism, was purified and characterized to homogeneity from skeletal muscle of Camelus dromedarius. The protein was purified approximately 26.8 folds by conventional ammonium sulphate fractionation followed by Blue Sepharose CL-6B chromatography, and its physical and kinetic properties were investigated. The native protein is a homotetramer with an apparent molecular weight of approximately 146 kDa. Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectric point of 7.2. The optimum pH of the purified enzyme was 7.8. Studies on the effect of temperature on enzyme activity revealed an optimal value of approximately 28-32 degrees with activation energy of 4.9 kcal/mol. The apparent K(m) values for NAD(+) and DL-glyceraldehyde-3-phophate were estimated to be 0.025+/-0.040 mM and 0.21+/-0.08 mM, respectively. The V(max) of the purified protein was estimated to be 52.7+/-5.9 U/mg. These kinetic parameter values were different from those described previously, reflecting protein differences between species.

Preferential Localization of Rat GAPDS on the Ribs of Fibrous Sheath of Sperm Flagellum and Its Expression during Flagellar Formation

The proper assembly of sperm flagellar proteins is fundamental for sperm motility. The sperm- and spermatid-specific isoform of glyceraldehyde 3-phosphate dehydrogenase, GAPDS, is a flagellar protein indispensable for sperm flagellar movement. To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody. Immunolocalization of rat GAPDS was restricted to the fibrous sheath (FS) in the sperm flagellum, and was predominant in the circumferential ribs rather than the longitudinal columns. Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation. Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

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 A(2)B(2)-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A(4)-GAPDH. GapB subunits of A(2)B(2)-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 A(2)B(2)-GAPDH into strongly inactivated A(8)B(8)-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 A(4)-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A(2)B(2)-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

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.