Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3.

Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu(2+), which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca(2+)-dependent, and CaM-binding domain is localized to 35-70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca(2+) signaling and cyclophilin activity in Arabidopsis.

Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin-binding isoform.

Calmodulin is a regulatory protein activated during Ca2+ signalling. We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin-binding diacylglycerol kinase) encoding a novel calmodulin-binding protein with sequence similarity to diacylglycerol kinases from animals. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid. We delineated the calmodulin-binding domain to approximately 25 residues near the C-terminus of LeCBDGK. We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin-binding domain. Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro. Anti-DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions. The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin-agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin-binding protein. In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions. Native LeCBDGK and calmodulin co-immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo. The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin-binding domain of LeCBDGK is encoded by a separate exon. Thus, alternative transcript splicing leads to calmodulin-binding and non-binding forms of diacylglycerol kinases in tomato. Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.

Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.

Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis.

Agricultural productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. Overexpression of a vacuolar Na+/H+ antiport from Arabidopsis thaliana in Arabidopsis plants promotes sustained growth and development in soil watered with up to 200 millimolar sodium chloride. This salinity tolerance was correlated with higher-than-normal levels of AtNHX1 transcripts, protein, and vacuolar Na+/H+ (sodium/proton) antiport activity. These results demonstrate the feasibility of engineering salt tolerance in plants.

Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution.

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

Activation of a plant plasma membrane Ca2+ channel by TGalpha1, a heterotrimeric G protein alpha-subunit homologue.

Wild-type and GTPase-deficient recombinant TGalpha1 were used along patch-clamp techniques to study the role of heterotrimeric G proteins in the regulation of the hyperpolarized active tomato plasma membrane Ca2+ channel. Recombinant alpha-subunits induced an increase in channel activity as shown by the increase in channel events and the mean open probability of the channel. Our results suggest a membrane-delimited pathway involving heterotrimeric G proteins in Ca2+ channel activation.

Characterization of the plant homologue of prohibitin, a gene associated with antiproliferative activity in mammalian cells.

This report describes the cloning and characterization of a plant cDNA coding for a protein which shows high amino acid sequence similarity with prohibitin, whose gene is associated with antiproliferative activity in mammalian cells. Arabidopsis thaliana and Nicotiana tabacum prohibitin complete cDNAs were isolated, and the expression pattern of prohibitin was examined using polyclonal antibodies raised against the Arabidopsis recombinant prohibitin expressed in Escherichia coli. A single immunoreactive protein was detected in various plant species and in all Arabidopsis organs examined. Subcellular fractionation using tobacco leaves revealed prohibitin in a mitochondrial-enriched fraction. Phylogenetic conservation of prohibitin's amino acid sequence and subcellular localization suggests a similar function in plants, yeast and mammals.

Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain.

To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.

Calcium/Calmodulin Activation of Soybean Glutamate Decarboxylase.

Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-amide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 [mu]M and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.

Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme.

The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway.

Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells.

Addition of l-[U-(14)C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-(14)C]glutamate, and (c) efflux of [(14)C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H(+)/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H(+)/l-Glu symport may involve both H(+) consumption during 4-aminobutyrate production and ATP-driven H(+) efflux.