The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.

Aquatic photosynthesis is responsible for about half of the global production and is undertaken by a huge phylogenetic diversity of algae that are poorly studied. The diversity of redox-regulation of phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was investigated in a wide range of algal groups under standard conditions. Redox-regulation of PRK was greatest in chlorophytes, low or absent in a red alga and most chromalveolates, and linked to the number of amino acids between two regulatory cysteine residues. GAPDH regulation was not strongly-related to the different forms of this enzyme and was less variable than for PRK. Addition of recombinant CP12, a protein that forms a complex with PRK and GAPDH, to crude extracts inhibited GAPDH and PRK inversely in the Plantae, but in most chromalveolates had little effect on GAPDH and inhibited or stimulated PRK depending on the species. Patterns of enzyme regulation were used to produce a phylogenetic tree in which cryptophytes and haptophytes, at the base of the chromalveolates, formed a distinct clade. A second clade comprised only chromalveolates. A third clade comprised a mixture of Plantae, an excavate and three chromalveolates: a marine diatom and two others (a xanthophyte and eustigmatophyte) that are distinguished by a low content of chlorophyll c and a lack of fucoxanthin. Regulation of both enzymes was greater in freshwater than in marine taxa, possibly because most freshwaters are more dynamic than oceans. This work highlights the importance of understanding enzyme regulation in diverse algae if their ecology and productivity is to be understood.

A4 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purified from the green alga Chlamydomonas reinhardtii and was also overexpressed in Escherichia coli. Both purified A4 tetramers of recombinant and native GAPDH were characterized for the first time. The pH optimum for both native and recombinant enzymes was close to 7.8. The pKs of the residues involved in catalysis indicate that a cysteine and a histidine may take part in catalysis by chloroplast GAPDH, as is the case for glycolytic GAPDH. Native and recombinant GAPDH show Michaelis-Menten kinetics with respect to their cofactors, NADH and NADPH, with greater specificity for NADPH. The kinetic parameters are similar to those of the heterotetrameric A2B2 spinach chloroplast GAPDH. Native C. reinhardtii and recombinant GAPDHs exhibit a cooperative behavior towards the substrate 1,3-bisphosphoglycerate (BPGA). This positive cooperativity is specific to the C. reinhardtii enzyme, as higher plant A2B2 GAPDHs show Michaelis-Menten kinetics. Native GAPDH has twofold lower catalytic constant and K0.5 for BPGA than recombinant GAPDH. Mass spectrometry analysis of native GAPDH shows that it is a complex of GAPDH and the small protein CP12. In vitro reconstitution assays indicate that the kinetic differences are the result conformation changes of GAPDH upon association with CP12.

Two cold-passaged mutant vaccine viruses (cp12 and cp45) derived from the JS wild-type (wt) strain of human parainfluenza virus type 3 (PIV3) have been sequenced. These mutant viruses display the cold-adapted (ca), temperature-sensitive (ts), and attenuation (att) phenotypes. Sequence data indicate that both cp12 and cp45 sustained nucleotide substitutions during cold passage and subsequent cloning. Fifteen nucleotide changes were present in cp12 and 18 in cp45. Of these changes, some were present in the sequence of the prototype wt strain (Wash/47885/57) or were non-coding changes present in the open reading frames (ORFs). These were considered unlikely to be of significance in contributing to phenotypic differences between the mutants and the JS wt. There were nine remaining changes in cp12 and eight in cp45 that would most likely contribute to their phenotypes. For cp12, two were non-coding changes in regulatory regions, one in the 3' genome leader and one in the NP gene transcription start signal. The remaining seven changes resulted in amino acid substitutions in NP, F, HN, and L. For cp45, two mutations were in a non-coding regulatory region, the 3' genome leader. The remaining six changes resulted in amino acid substitutions in F, HN, and L. Only one amino acid substitution was conserved between cp12 and cp45 (a valine to alanine change at position 384 of the HN gene). These results should prove useful in the future in understanding the genetic basis of attenuation of the cold-passaged PIV3 candidate vaccine viruses.

Parainfluenza virus type 3 (para 3) was adapted to replicate at 20 degrees C, a nonpermissive temperature for wild-type (wt) para 3. Serial passage at 20 degrees C resulted in the generation of cold-adapted (ca) and temperature-sensitive (ts) mutants. These mutant viruses have been characterized both in vitro and in vivo [Belshe and Hissom (1982): Journal of Medical Virology 10:235-242; Crookshanks and Belshe (1984): Journal of Medical Virology 13:243-249]. We now report the evaluation of three mutants (clone 1150, passaged 12 times in the cold [cp12], clone 1146, passaged 18 times in the cold [cp18], and clone 1328, passaged 45 times in the cold [cp45]) for their ability to protect hamsters from infection by wild-type para 3. Ether-anesthetized male syrian hamsters were intranasally vaccinated with either wt para 3 (clone 127) or one of the ca para 3 mutants and on day 28 post-vaccination; each animal was intranasally challenged with 10(5.0) pfu of wt para 3. On days 1, 2, 3, and 4 post-challenge, 4 to 13 hamsters from each group were sacrificed, and the quantity of para 3 in the nasal turbinates and lungs was determined. Wt virus induced protection from challenge. cp12, cp18, and cp45 reduced the peak titer of wt replication in the lungs by greater than 100-fold, tenfold, and tenfold, respectively. The duration of virus replication was shortened also by intranasal vaccination with the mutants. These data give evidence of an inverse relationship between the degree of protection induced by vaccination with cold-adapted mutants and the number of passages of the virus in the cold.

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.

The chloroplast protein CP12 is known to play a leading role in a complex formation with the enzymes GAPDH and PRK. As a preliminary step towards the understanding of the complex formation mechanism and the exact role of this protein linker, a comparative modelling of the CP12 protein of the green alga Chlamydomonas reinhardtii was performed. Because of the very few structural information and poor template similarities, the derivation of the model consisted in an iterative trial-and-error procedure using the comparative modelling program MODELLER, the following three structure validation programs PROCHECK, PROSA, and WHATIF, and molecular mechanics energy refinement of the model using the program CHARMM. The analysis of the final model reveals a scaffold of key residues that is believed to be essential in the folding mechanism and that coincides with the residues conserved throughout the CP12 family. Our results suggest that this protein is a typical disordered protein. Finally, the various mechanisms by which the CP12 protein can self-interact or binds to other enzymes are discussed in light of its modelled structure and characteristics.

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.

A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A(4) glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific "chaperone-like protein" for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A(2)B(2) GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.

The chloroplast protein CP12 has been shown to regulate the activity of two Calvin cycle enzymes, phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by the reversible formation of a multiprotein complex. In Arabidopsis there are three CP12 genes, CP12-1, CP12-2, and CP12-3, and expression analysis suggested that the function of these proteins may not be restricted to the Calvin cycle. Reverse transcription-PCR analysis was used here to investigate further the expression patterns of the three CP12 Arabidopsis genes together with the genes encoding plastid GAPDH (GAPA-1 and GAPB), PRK (PRK), and plastid NAD-dependent GAPDH (GAPCp1 and GAPCp2) during development, in response to changes in light, temperature, and anaerobic conditions. Expression of the CP12-2 gene was similar to that of the Calvin cycle enzymes PRK and GAPDH. However, this was not the case for CP12-1 and -3 which were both expressed in roots. Analysis of transgenic Arabidopsis lines expressing CP12::GUS fusion constructs revealed that the CP12 genes display different spatiotemporal expression patterns. The CP12-1 gene was expressed in root tips whilst CP12-3::GUS expression was evident throughout the root tissue. The most unexpected finding was that all three CP12 genes were expressed in floral tissues; CP12-1 and CP12-2 expression was detected in the sepals and the style of the flower, while in contrast CP12-3::GUS expression was restricted to the stigma and anthers. Taken together, the data suggest that the redox-sensitive CP12 proteins may have a wider role in non-photosynthetic plastids, throughout the plant life cycle.

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.

In oxygenic photosynthetic organisms, the activities of two Calvin cycle enzymes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH and phosphoribulokinase, PRK) are regulated by CP12-mediated complex formation. The Arabidopsis genome contains three genes encoding different CP12 isoforms (CP12-1, At2g47400; CP12-2, At3g62410 and CP12-3, At1g76560), all plastid-targeted, as demonstrated by localization in the chloroplast stroma of CP12 precursor sequences fused with the green fluorescence protein (GFP). The disorder predictor PONDR classified Arabidopsis CP12s as largely disordered proteins, and circular dichroism spectra confirmed these predictions. Based on sequence similarity, 66 CP12s from different organisms were identified and clustered in six types, with CP12-1 and -2 grouping together with other largely disordered sequences (Type I), while a lower level of disorder was predicted within the cluster including CP12-3 (Type II). The three Arabidopsis CP12 isoforms were expressed as mature recombinant forms and purified to homogeneity. Redox titrations demonstrated that the four conserved cysteines of each CP12 isoform could form two internal disulfide bridges with different midpoint redox potentials (E(m,7.9) -326 mV and -350 mV in both CP12-1 and CP12-2; E(m,7.9) -332 mV and -373 mV in CP12-3). In agreement with their similar redox properties, all CP12 isoforms formed, in vitro, a supramolecular complex with GAPDH and PRK, with comparable inhibitory effects on both enzyme activities. In order to test whether CP12 isoforms might have broader regulatory functions than regulating Calvin cycle enzymes, CP12 proteins were analyzed for their capacity to bind plastidial glycolytic GAPDH (GapCp). To this purpose, the mature form of Arabidopsis GapCp2 was cloned, expressed in recombinant form and purified to homogeneity. However, contrary to expectations, no CP12 isoform was able to bind GapCp2 under any of the conditions tested.

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.

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin-Benson cycle, whose regulation is crucial for the global balance of the photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin-Benson cycle from oxidative stress.

Glyceraldehyde-3-phosphate dehydrogenase (GapAB) and CP12 are two major players in controlling the inactivation of the Calvin cycle in land plants at night. GapB originated from a GapA gene duplication and differs from GapA by the presence of a specific C-terminal extension that was recruited from CP12. While GapA and CP12 are assumed to be generally present in the Plantae (glaucophytes, red and green algae, and plants), up to now GapB was exclusively found in Streptophyta, including the enigmatic green alga Mesostigma viride. However, here we show that two closely related prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, also possess a GapB gene, while CP12 is missing. This remarkable finding either antedates the GapA/B gene duplication or indicates a lateral recruitment. Moreover, Ostreococcus is the first case where the crucial CP12 function may be completely replaced by GapB-mediated GapA/B aggregation.

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.

CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12-Cu(2+) complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp(16)-Asp(23), Asp(38)-Lys(50) and Asp(70)-Glu(76), with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.

Monoclonal antibodies (MAbs) were produced to Brucella melitensis cytosoluble proteins (CP) with apparent molecular masses of 12, 24 and 28 kDa (CP12, CP24 and CP28) which were previously shown by immunoblotting to differentiate antibody responses of infected sheep from those of B. melitensis strain Rev. 1 vaccinated sheep. These MAbs were derived from mice infected with virulent smooth (S) B. melitensis strain H38. Most MAbs obtained were directed to CP28, which indicated (as was shown in infected sheep) that this protein was also highly immunogenic in mice. A large number of MAbs that showed reactivity to CP in ELISA but did not show reactivity in immunoblotting of CP were also obtained and might recognise conformational epitopes of these proteins. MAbs were used to localise CP12, CP24 and CP28. None of the MAbs reacted with whole B. melitensis cells in ELISA but showed reactivity with sonicated bacteria in ELISA, which indicated an internal localisation of these proteins. Among several B. melitensis B115 subcellular fractions tested, the anti-CP12 and anti-CP28 MAbs reacted essentially with the CP extract (CPE) in both ELISA and immunoblotting, whereas the anti-CP24 MAbs reacted with both CPE and cell envelope fraction (CEF)-although with lower intensity to the latter fraction. The internal localisation of these proteins was confirmed by immuno-electron microscopy of thin-sectioned B. melitensis B115 or B. melitensis 16M cells. Immunogold labelling was mainly observed in the cytoplasm and, consequently, CP12, CP24 and CP28 are probably cytoplasmic proteins. Immunoblotting of whole cell lysates with the MAbs also showed the presence of these proteins in all Brucella species and biovars, including the vaccine strains B. melitensis Rev. 1 and B. abortus B19. The use of these MAbs should help further study of antibody responses in sheep and other hosts and may be of considerable value for developing new diagnostic tests for ovine brucellosis.

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.

CP12 is a flexible protein that is well-known to interact with GAPDH, and this association is crucial to the regulation of enzyme activity. This regulation is likely related to structural transitions of both proteins, but the molecular bases of these changes are not yet understood. To answer this issue, we undertook a study based on the use of paramagnetic probes grafted on cysteine residues and followed by EPR spectroscopy. We present a new application of this approach that enables us to probe the functional role of cysteine residues in protein-protein interactions. Algal CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state and has some alpha-helical secondary structural elements. In contrast, in its reduced state, CP12 is mainly unstructured and shares some physical properties with intrinsically disordered proteins. Treatment of CP12 with a methane thiosulfonate derivative spin-label (MTSL) led to the labeling of the cysteine residues involved in the C-terminal bridge only as revealed by mass spectrometry. Surprisingly, the partner protein GAPDH induced the cleavage of the disulfide bridge between the cysteine residues of CP12 and the spin-label, resulting in the full release of the label. We showed the existence of a transitory interaction between both proteins and proposed a mechanism based on a thiol-disulfide exchange reaction. The results of this study point out a novel role of the algal GAPDH which is often termed a "moonlighting" protein.

UNASSIGNED

Alfalfa (Medicago sativa) is the most extensively cultivated forage legume in the world, and salinity stress is the most problematic environmental factors limiting alfalfa production. To evaluate alfalfa tissue variations in response to salt stress, comparative physiological and proteomic analyses were made of salt responses in the roots and shoots of the alfalfa.

UNASSIGNED

A two-dimensional gel electrophoresis (2-DE)-based proteomic technique was employed to identify the differentially abundant proteins (DAPs) from salt-treated alfalfa roots and shoots of the salt tolerance cultivars Zhongmu No 1 cultivar, which was subjected to a range of salt stress concentrations for 9 days. In parallel, REL, MAD and H2O2 contents, and the activities of antioxidant enzymes of shoots and roots were determinand.

UNASSIGNED

Twenty-seven spots in the shoots and 36 spots in the roots that exhibited showed significant abundance variations were identified by MALDI-TOF-TOF MS. These DAPs are mainly involved in the biological processes of photosynthesis, stress and defense, carbohydrate and energy metabolism, second metabolism, protein metabolism, transcriptional regulation, cell wall and cytoskeleton metabolism, ion transpor, signal transduction. In parallel, physiological data were correlated well with our proteomic results. It is worth emphasizing that some novel salt-responsive proteins were identified, such as CP12, pathogenesis-related protein 2, harvest-induced protein, isoliquiritigenin 2'-O-methyltransferase. qRT-PCR was used to study the gene expression levels of the four above-mentioned proteins; four patterns are consistent with those of induced protein.

UNASSIGNED

The primary mechanisms underlying the ability of alfalfa seedlings to tolerate salt stress were photosynthesis, detoxifying and antioxidant, secondary metabolism, and ion transport. And it also suggests that the different tissues responded to salt-stress in different ways.

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

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.