Regulation of starch accumulation by granule-associated plant 14-3-3 proteins.

In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins participate in environmentally responsive phosphorylation-related regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4-fold increase in leaf starch accumulation. Dark-governed starch breakdown was unaffected in these "antisense plants," indicating an unaltered starch-degradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indicate that the starch from the antisense plants has an altered branched glucan composition. Biochemical characterization of protease-treated starch granules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 proteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III family is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, all members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif. This possibility is strengthened by immunocapture using antibodies to DU1, a maize starch synthase III family member, and direct interaction with biotinylated 14-3-3 protein, both of which demonstrated an association between 14-3-3 proteins and DU1 or DU1-like proteins.

Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma.

The 14-3-3 proteins are acidic, dimeric proteins that have been implicated in many eukaryotic cellular processes because of direct protein association with enzymes and other metabolic and regulatory proteins. 14-3-3 proteins are largely considered to be cytoplasmic, but a search for proteins that specifically interact with a plant 14-3-3 resulted in the isolation of a nuclear-encoded, thylakoid-targeted chloroplast precursor, the full-length Arabidopsis photosystem I N-subunit At pPSI-N (P.C. Sehnke, R.J. Ferl ¿1995 Plant Physiol 109: 1126). Using precursor truncations in the two-hybrid system, it was determined that the leader sequence is the site of PSI-N that associates with 14-3-3. This suggested the novel possibility that 14-3-3 would be found within chloroplasts. Immuno-electron microscopy of leaf tissue and western analysis of chloroplast fractions with monoclonal anti-14-3-3 antibodies localized 14-3-3 proteins to the chloroplast stroma and the stromal side of thylakoid membranes. Using peptide-generated, isoform-specific antibodies, GF14nu, GF14epsilon, GF14mu, and GF14upsilon were shown to be present in the chloroplast stromal extract. These isoforms represent two distinct phylogenetic 14-3-3 groupings. These data suggest a novel interorganellar role for these phylogenetically distinct 14-3-3 proteins.

Plant 14-3-3s: omnipotent metabolic phosphopartners?

The accurate regulation of metabolism is crucial to the existence all organisms. The inappropriate activation of metabolic enzymes can waste precious energy; likewise, the failure to activate metabolic enzymes can disrupt homeostasis and lead to suboptimal cellular (and organismic) responses. Plants use several means to control their metabolic proteins, including a two-step process of protein phosphorylation and subsequent binding by phosphospecific binding proteins termed 14-3-3 proteins. Sehnke and Ferl discuss how 14-3-3 proteins regulate the activity of nitrate reductase and the H(+)-ATPase pump in plants, and compare the functions of 14-3-3 proteins in plants and animals.

Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex.

The 14-3-3 family of multifunctional proteins is highly conserved among animals, plants, and yeast. Several studies have shown that these proteins are associated with a G-box DNA binding complex and are present in the nucleus in several plant and animal species. In this study, 14-3-3 proteins are shown to bind the TATA box binding protein (TBP), transcription factor IIB (TFIIB), and the human TBP-associated factor hTAF(II)32 in vitro but not hTAF(II)55. The interactions with TBP and TFIIB were highly specific, requiring amino acid residues in the box 1 domain of the 14-3-3 protein. These interactions do not require formation of the 14-3-3 dimer and are not dependent on known 14-3-3 recognition motifs containing phosphoserine. The 14-3-3-TFIIB interaction appears to occur within the same domain of TFIIB that binds the human herpes simplex virus transcriptional activator VP16, because VP16 and 14-3-3 were able to compete for interaction with TFIIB in vitro. In a plant transient expression system, 14-3-3 was able to activate GAL4-dependent beta-glucuronidase reporter gene expression at low levels when translationally fused with the GAL4 DNA binding domain. The in vitro binding with general transcription factors TBP and TFIIB together with its nuclear location provide evidence supporting a role for 14-3-3 proteins as transcriptional activators or coactivators when part of a DNA binding complex.

Processing of preproricin in transgenic tobacco.

The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis. Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter. Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques. Coomassie blue staining of an SDS-PAGE gel of lactose-agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight. Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin. High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol. N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide. Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin.

A novel nuclear member of the thioredoxin superfamily.

We describe the isolation and characterization of a cDNA encoding maize (Zea mays L.) nucleoredoxin (NRX), a novel nuclear protein that is a member of the thioredoxin (TRX) superfamily. NRX is composed of three TRX-like modules arranged as direct repeats of the classic TRX domain. The first and third modules contain the amino acid sequence WCPPC, which indicates the potential for TRX oxidoreductase activity, and insulin reduction assays indicate that at least the third module possesses TRX enzymatic activity. The carboxy terminus of NRX is a non-TRX module that possesses C residues in the proper sequence context to form a Zn finger. Immunolocalization preferentially to the nucleus within developing maize kernels suggests a potential for directed alteration of the reduction state of transcription factors as part of the events and pathways that regulate gene transcription.

The heterologous interactions among plant 14-3-3 proteins and identification of regions that are important for dimerization.

The 14-3-3 proteins constitute a family of dimeric proteins that are involved in many cellular functions. At least two mammalian 14-3-3 proteins can form heterodimers and the approximate regions important for dimerization have been identified. In this study, we demonstrate that eight Arabidopsis and one maize 14-3-3 protein can dimerize with each other and with themselves. Native gel Western analysis of Arabidopsis cell extract also suggests the presence of 14-3-3 heterodimers in vivo. Finally, we identified the domains of one 14-3-3 protein that are sufficient for homodimerization and heterodimerization. These data support the hypothesis that evolutionarily divergent 14-3-3 proteins can interact with each other to form diverse molecular modulators or adapters in signaling pathways.

Plant metabolism: enzyme regulation by 14-3-3 proteins.

14-3-3 proteins have been found to regulate the plant enzyme nitrate reductase by reversible phosphoserine binding. Plant plasma-membrane H(+)-ATPases, transporters that are activated by the phytotoxin fusicoccin, appear to be regulated in a similar fashion.

A chromatographic analysis of capsid protein isolated from alfalfa mosaic virus: zinc binding and proteolysis cause distinct charge heterogeneity.

The capsid protein (CP) of alfalfa mosaic virus (AIMV) is required for viral replication when susceptible plants are inoculated with purified viral genomic RNA. The discovery of AIMV CP in the zinc activated RNA-dependent RNA polymerase complex prompted our further investigation of AIMV virions and the potential involvement of AIMV CP in metal binding. AIMV CP, isolated from nucleoprotein components, fractionated into four distinct ionic species when purified by cation exchange fast protein liquid chromatography. The CP existed as zinc complexed homodimers, metal-free homodimers, and two forms of proteolyzed heterodimers, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography, amino-terminal sequencing, and atomic absorption spectroscopy. Although the relative amounts of proteolyzed heterodimers varied, the ratio of zinc complexed homodimers to metal-free homodimers (1:10) was constant between virus and protein isolations for the strains 425 and WISC14. Purified metal-free and zinc-complexed homodimers could be interconverted in vitro by incubation with zinc chloride or with the metal chelator, sodium diethyldithiocarbamate (NaDDC). The potential role of zinc in AIMV nucleoprotein structure and infectivity was investigated by treatment of the virions with NaDDC. Electron microscopy and sucrose density gradient studies failed to detect any gross structural changes for zinc depleted virus; however, a decrease in infectivity was observed with local lesion leaf assays, suggesting a functional role for zinc in viral replication.

Expression of active, processed ricin in transgenic tobacco.

The cDNA encoding the plant toxin precursor preproricin was introduced into tobacco via Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants were assayed for type II ribosome-inactivating protein expression and activity. Western blot analysis of soluble leaf extracts using anti-ricin a-chain (RTA) antibodies identified 34- and 32-kDa proteins, which were electrophoretically indistinguishable from castor seed RTA. Analysis with anti-ricin b-chain (RTB) antibodies identified both a 34-kDa protein major band, which co-migrated with castor seed RTB, and a 30-kDa protein minor band. Enzyme-linked immunoassay of the transgenic leaf extracts with anti-RTA and anti-RTB indicated microgram per gram production on a fresh weight basis of soluble extractable recombinant ricin. Sugar binding enzyme-linked immunoassay employing an immobilized glycoprotein, asialofetuin, and anti-RTB antibodies confirmed the characteristic type II ribosome-inactivating protein galactose binding lectin activity of the recombinant ricin. The enzymatic activity of recombinant ricin was characterized for cell-free translation inhibition, as well as for overall cytotoxicity. A 50% inhibitory dose of 3 x 10(-11) M was observed for the immunoreactive leaf extract material using a rabbit reticulocyte translation inhibition assay, while a 50% lethal dose of 1 x 10(-12) M was calculated with human T-lymphotropic virus-1 infected leukemic T-cells.

A single Arabidopsis GF14 isoform possesses biochemical characteristics of diverse 14-3-3 homologues.

Arabidopsis cDNA clones of GF14 proteins originally were isolated on the basis of their association with the G-box DNA/protein complex by a monoclonal antibody screening approach. GF14 proteins are homologous to the 14-3-3 family of mammalian proteins. Here we demonstrate that recombinant GF14 omega, one member of the Arabidopsis GF14 protein family, is a dimeric protein that possesses many of the attributes of diverse mammalian 14-3-3 homologues. GF14 omega activates rat brain tryptophan hydroxylase and protein kinase C in a manner similar to the bovine 14-3-3 protein. It also activates exoenzyme S of Pseudomonas aeruginosa as does bovine brain factor activating exoenzyme S (FAS), which is itself a member of 14-3-3 proteins. In addition, GF14 omega binds calcium, as does the human 14-3-3 homologue reported to be a phospholipase A2. These results indicate that a single isoform of this plant protein family can have multiple functions and that individual GF14 isoforms may have multiple roles in mediating signal transductions in plants. However, GF14 omega does not regulate growth in an in vivo test for functional similarity to the yeast 14-3-3 homologue, BMH1. Thus, while a single plant GF14 isoform can exhibit many of the biochemical attributes of diverse mammalian 14-3-3 homologues, open questions remain regarding the physiological functions of GF14/14-3-3 proteins.

Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog.

Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes. Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer. Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium. Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions. The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding. The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody. In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro. Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants. The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.

Crystallization and preliminary X-ray characterization of tobacco streak virus and a proteolytically modified form of the capsid protein.

Isolated tobacco streak virus strain mild (TSV-M) top nucleoprotein component (TV) was crystallized by vapor diffusion with polyethylene glycol (PEG) and methyl pentanediol. The morphology of the crystal suggested a hexagonal space group, however, the crystals were disordered as analyzed by X-ray diffraction. Capsid protein from TSV (strains M and white clover) was treated with trypsin to remove 87 amino terminal residues. The modified capsid protein was purified by ion exchange chromatography and crystallized in both hexagonal and triclinic forms. Hexagonal crystals grown by vapor diffusion in PEG diffract X rays to 4.5 A. The crystals, in the space group P6(2) or its enantiomorph, possess unit cell dimensions of a = 98.3 A, c = 108.7 A and probably contain 12 dimers/unit cell. Triclinic crystals grown by vapor diffusion in ammonium sulfate diffracted X rays to 2.4 A resolution when exposed to X-ray synchrotron radiation. The crystals have unit cell dimensions of a = 60.1 A, b = 77.5 A, c = 73.9 A with alpha = 67.8 degrees, beta = 130 degrees, and gamma = 106 degrees and probably contain 4 dimers/unit cell.

A "zinc-finger"-type binding domain in tobacco streak virus coat protein.

Tobacco streak virus (TSV) RNA and alfalfa mosaic virus (AIMV) RNA will replicate only if a few copies of their coat proteins are bound to the RNA. To understand this phenomenon experiments were performed to find unique features of the TSV and AIMV subunits. Atomic absorption analysis showed that TSV and AIMV contained substantial quantities of zinc in native virions (approximately one zinc atom per four protein subunits in TSV and one zinc atom per two protein subunits in AIMV), while other plant viruses tested did not. Treatment of TSV with a zinc-extracting reagent resulted in partial degradation of all the TSV nucleoprotein components, although the top component was most effected. The sequence (Cys X2 Cys X10 Cys X2 His) was found between residues 28 and 45 in the TSV primary structure and it is similar to a sequence found in several nucleic acid-binding, gene-regulatory proteins, most notably transcription factor IIIA from Xenopus laevis. TSV subunits were found to be extensively crosslinked within the virions. TSV and AIMV contain sequences rich in basic residues in the amino-terminal portion of the subunit (residues 51 to 72 in TSV and 1 to 26 in AIMV) and helical predictions suggested modes of protein-nucleic acid interactions in these regions similar to those proposed for histones. Two potential sites for glycosylation were identified near the amino terminus of the TSV sequence. Controlled treatment of TSV with trypsin removed 87 residues from the amino terminus and produced a monomer of cleaved protein, as analysed by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)