In vitro co-expression chromatin assembly and remodeling platform for plant histone variants.

Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A.

De novo reconstitution of chromatin using wheat germ cell-free protein synthesis.

DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell-free protein synthesis.

Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system.

Background: Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins.

A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression.

Background: Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively.

Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting.

Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6.

Coevolving MAPK and PID phosphosites indicate an ancient environmental control of PIN auxin transporters in land plants.

Plant growth flexibly adapts to environmental conditions, implying cross-talk between environmental signalling and developmental regulation. Here, we show that the PIN auxin efflux carrier family possesses three highly conserved putative mitogen-activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well-characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6.

Kinase-Associated Phosphoisoform Assay: a novel candidate-based method to detect specific kinase-substrate phosphorylation interactions in vivo.

Background: Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo.

Glucose transporter type 10-lacking in arterial tortuosity syndrome-facilitates dehydroascorbic acid transport.

Loss-of-function mutations in the gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder. In this study GLUT10-mediated dehydroascorbic acid (DAA) transport was investigated, supposing its involvement in the pathomechanism. GLUT10 protein produced by in vitro translation and incorporated into liposomes efficiently transported DAA.

The Arabidopsis mitogen-activated protein kinase 6 is associated with γ-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress.

Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro.

Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Mitogen-activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organized into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The Arabidopsis mitogen-activated protein kinase 9 (AtMPK9) is a member of an atypical class of MAPKs.

A rational approach for generating cardiac troponin I selective Spiegelmers.

We report the first protein selective Spiegelmers of diagnostic relevance by rational identification of a target epitope and reverse screening of Spiegelmer candidates following the selection procedure. Application of the presented approach resulted in isolation of cardiac troponin I selective Spiegelmers with low nanomolar dissociation constant and functionality in serum.

In vitro translation-based protein kinase substrate identification.

Identification of a particular protein as a physiological substrate towards kinases of interest is an extremely complex process. Under physiological conditions kinases and their putative substrates are in low abundance, and production of active eukaryotic kinases with standard overexpressing methods is an arduous task. Herein, we describe a cell-free in vitro protein translation procedure combined with fluorescent phosphoprotein staining as a simple and rapid method for identification of putative kinase substrates.