The Acidic A-Domain of Arabidopsis Toc159 Occurs as a Hyperphosphorylated Protein

The translocon at the outer membrane of the chloroplast assists the import of a large class of preproteins with amino-terminal transit sequences. The preprotein receptors Toc159 and Toc33 in Arabidopsis (Arabidopsis thaliana) are specific for the accumulation of abundant photosynthetic proteins. The receptors are homologous GTPases known to be regulated by phosphorylation within their GTP-binding domains. In addition to the central GTP-binding domain, Toc159 has an acidic N-terminal domain (A-domain) and a C-terminal membrane-anchoring domain (M-domain). The A-domain of Toc159 is dispensable for its in vivo activity in Arabidopsis and prone to degradation in pea (Pisum sativum). Therefore, it has been suggested to have a regulatory function. Here, we show that in Arabidopsis, the A-domain is not simply degraded but that it accumulates as a soluble, phosphorylated protein separated from Toc159. However, the physiological relevance of this process is unclear. The data show that the A-domain of Toc159 as well as those of its homologs Toc132 and Toc120 are targets of a casein kinase 2-like activity.The Toc and Tic complexes cooperate to import nuclear-encoded chloroplast preproteins from the cytosol (Jarvis, 2008; Kessler and Schnell, 2009). Initially, incoming preproteins encounter the receptors Toc159 and Toc34 at the chloroplast surface. Both are GTP-binding proteins and share sequence homology in their G-domains. While Toc34 is anchored in the outer membrane by a short hydrophobic C-terminal tail, the triple-domain Toc159 is inserted via a largely hydrophilic 52-kD M-domain. In addition to the G- and M-domains, Toc159 has a large acidic A-domain covering the N-terminal half of the protein. Arabidopsis (Arabidopsis thaliana) encodes two isoforms of Toc34 (Toc33 and Toc34) and four of Toc159 (Toc159, Toc132, Toc120, and Toc90; Jackson-Constan and Keegstra, 2001). The Toc159 isoforms have a similar domain structure, but they differ from each other in length and sequence of their A-domain (Hiltbrunner et al., 2001a). However, Toc90 does not have an acidic domain at all and only consists of the G- and M-domains (Hiltbrunner et al., 2004). It has been demonstrated that the A-domain of AtToc159 and AtToc132 have properties of intrinsically disordered proteins (Hernández Torres et al., 2007; Richardson et al., 2009), suggesting an involvement of the A-domain in transient and multiple protein-protein interactions possibly with the transit peptides of preproteins. Toc34 and Toc159 together with the Toc75 channel constitute the Toc-core complex (Schleiff et al., 2003) and are required for the accumulation of highly abundant photosynthesis-associated proteins in the chloroplast. The Arabidopsis deletion mutants of Toc33 (ppi1; Jarvis et al., 1998) and Toc159 (ppi2; Bauer et al., 2000) have indicative phenotypes of their role in chloroplast biogenesis, respectively pale green and albino. Complementation experiments of the ppi2 mutant have established that the G- and M-domains have essential functions whereas the A-domain is dispensable (Lee et al., 2003; Agne et al., 2009). In preceding studies, possibly influenced by the model organism and experimental tools, Toc159 occurred in different forms. Initially, Toc159 was identified in pea (Pisum sativum) as an 86-kD protein lacking the entire A-domain (Hirsch et al., 1994; Bolter et al., 1998). In addition to its membrane-associated form, Arabidopsis Toc159 has been found as a soluble protein (Hiltbrunner et al., 2001b). However, the function and the fate of the A-domain as well as that of soluble Toc159 remain unknown and a matter of debate.Not only GTP binding and hydrolysis by the Toc GTPases but also phosphorylation is known as a regulatory mechanism of chloroplast protein import at the Toc complex level (Oreb et al., 2008b). First, some precursor proteins, such as the small subunit of Rubisco, may be phosphorylated in their transit sequence by a cytosolic kinase (Martin et al., 2006). Phosphorylation promotes binding to a 14-3-3 protein and cytosolic Hsp70 in the guidance complex that delivers the phosphorylated preprotein to the Toc complex (May and Soll, 2000). Second, both Toc159 and Toc34 are known to be phosphorylated and independently so by distinct kinases, OEK70 and OEK98, respectively (Fulgosi and Soll, 2002). These two kinase activities have been located to the outer envelope membrane, but their molecular identification is still pending. Phosphorylation of the Toc GTPases may occur in the GTP-binding domains (Oreb et al., 2008a). For Toc34, data on the site (Ser-113 in pea and Ser-181 in Arabidopsis) and effects of phosphorylation are available (Jelic et al., 2002, 2003). It imposes a negative regulation on the Toc complex by inhibiting GTP and preprotein binding to Toc34, reducing its ability to bind Toc159 and to assemble into the Toc complex (Oreb et al., 2008a). The in vivo mutational analysis in Arabidopsis indicated that phosphorylation at Toc34 represents a nonessential mechanism (Aronsson et al., 2006; Oreb et al., 2007). Despite the 86-kD proteolytic fragment of Toc159 being a major phosphoprotein in the pea outer chloroplast membrane (Fulgosi and Soll, 2002), little is known of the molecular and regulatory mechanisms of Toc159 phosphorylation. In this study, we report that the A-domain of Toc159 can be purified as a stable fragment. Moreover, it is hyperphosphorylated, hinting at an important and highly regulated functional role. Our data suggest that Toc159 is the target of casein kinase 2 (CK2)-like and membrane-associated kinase activities.     

The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.     

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.     

The p53 proto-oncogene can act as a suppressor of transformation

DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.     

Copepods act as a switch between alternative trophic cascades in marine pelagic food webs

A recent meta‐analysis indicates that trophic cascades (indirect effects of predators on plants via herbivores) are weak in marine plankton in striking contrast to freshwater plankton ( Shurin et al. 2002 , Ecol. Lett., 5, 785–791). Here we show that in a marine plankton community consisting of jellyfish, calanoid copepods and algae, jellyfish predation consistently reduced copepods but produced two distinct, opposite responses of algal biomass. Calanoid copepods act as a switch between alternative trophic cascades along food chains of different length and with counteracting effects on algal biomass. Copepods reduced large algae but simultaneously promoted small algae by feeding on ciliates. The net effect of jellyfish on total algal biomass was positive when large algae were initially abundant in the phytoplankton, negative when small algae were dominant, but zero when experiments were analysed in combination. In contrast to marine systems, major pathways of energy flow in Daphnia‐dominated freshwater systems are of similar chain length. Thus, differences in the length of alternative, parallel food chains may explain the apparent discrepancy in trophic cascade strength between freshwater and marine planktonic systems.     

Subunits act independently in a cyclic nucleotide-activated K(+) channel

Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide-activated K(+) channel from Mesorhizobium loti and its isolated cyclic nucleotide-binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non-cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide-gated channels.     

CARPEL FACTORY,a Dicer homolog,and HEN1, a novel protein,act in microRNA metabolism in Arabidopsis thaliana

BACKGROUND: In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19-25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism.RESULTS: To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes.CONCLUSIONS: miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.     

Epigenetic mechanisms in the dentate gyrus act as a molecular switch in hippocampus-associated memory formation

We make strong memories of significant events in our lives which may serve to increase our resilience and adaptation capacity to deal with future challenges. It is well established that the neurotransmitter glutamate and the ERK MAPK intracellular signaling pathway play a principal role in memory formation. In addition, stress-associated hormones like glucocorticoids released during such events are known to strengthen formation of memories. But, how do these hormones work? Do they interact with the ERK MAPK pathway or otherwise? What are the more distal, epigenomic effects? We discovered in rats and mice that confrontation with a psychological challenge (e.g. forced swimming, Morris water maze) would lead, through NMDA-ERK signaling, to MSK1 and Elk-1 activation in dentate gyrus neurons (a part of the hippocampus involved in encoding of memories) resulting in histone H3 S10-phosphorylation and K14-acetylation, H4 hyper-acetylation, gene induction and formation of memories of the event. Moreover, glucocorticoid hormones via the glucocorticoid receptor (GR) greatly facilitated the epigenomic mechanisms and cognitive performance. Therefore, we propose that formation of enduring memories of significant events requires an interaction of GRs with the NMDA/ERK/MSK1/Elk-1 signaling pathways to allow an optimal epigenomic activation pattern in dentate gyrus neurons to accommodate their altered neurophysiological function.     

Ionic channel density of excitable membranes can act as a bifurcation parameter

As the maximal K⁺-conductance (or K⁺-channel density) of the Hodgkin-Huxley equations is reduced, the stable resting membrane potential bifurcates at a subcritical Hopf bifurcation into small amplitude unstable oscillations. These small amplitude solutions jump to large amplitude periodic solutions that correspond to a repetitive discharge of action potentials. Thus the specific channel density can act as a bifurcation parameter, and can control the excitability and autorhythmicity of excitable membranes.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators

APOBEC1 is the catalytic component of an RNA editing complex but shows homology to activation-induced cytidine deaminase (AID), a protein whose function is to potentiate diversification of immunoglobulin gene DNA. Here, we show that APOBEC1 and its homologs APOBEC3C and APOBEC3G exhibit potent DNA mutator activity in an E. coli assay. Indeed, like AID, these proteins appear to trigger DNA mutation through dC deamination. However, each protein exhibits a distinct local target sequence specificity. The results reveal the existence of a family of potential active dC/dG mutators, with possible implications for cancer.     

Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton

Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions.     

Biological evidence that SOCS-2 can act either as an enhancer or suppressor of growth hormone signaling

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.