In vivo homopropargylglycine incorporation enables sampling, isolation and characterization of nascent proteins from Arabidopsis thaliana.

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.

Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC.

The phosphate esters of myo-inositol (Ins) occur ubiquitously in biology. These molecules exist as soluble or membrane-resident derivatives and regulate a plethora of cellular functions including phosphate homeostasis, DNA repair, vesicle trafficking, metabolism, cell polarity, tip-directed growth, and membrane morphogenesis. Phosphorylation of all inositol hydroxyl groups generates phytic acid (InsP6), the most abundant inositol phosphate present in eukaryotic cells. However, phytic acid is not the most highly phosphorylated naturally occurring inositol phosphate. Specialized small molecule kinases catalyze the formation of the so-called myo-inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8. These molecules are characterized by one or several "high-energy" diphosphate moieties and are ubiquitous in eukaryotic cells. In plants, PP-InsPs play critical roles in immune responses and nutrient sensing. The detection of inositol derivatives in plants is challenging. This is particularly the case for inositol pyrophosphates because diphospho bonds are labile in plant cell extracts due to high amounts of acid phosphatase activity. We present two steady-state inositol labeling-based techniques coupled with strong anion exchange (SAX)-HPLC analyses that allow robust detection and quantification of soluble and membrane-resident inositol polyphosphates in plant extracts. These techniques will be instrumental to uncover the cellular and physiological processes controlled by these intriguing regulatory molecules in plants.

COMET Functions as a PCH2 Cofactor in Regulating the HORMA Domain Protein ASY1.

The formation of the chromosome axis is key to meiotic recombination and hence the correct distribution of chromosomes to meiotic products. A key component of the axis in Arabidopsis is the HORMA domain protein (HORMAD) ASY1, the homolog of Hop1 in yeast and HORMAD1/2 in mammals. The chromosomal association of ASY1 is dynamic, i.e., ASY1 is recruited to the axis at early prophase and later largely removed when homologous chromosomes synapse. PCH2/TRIP13 proteins are well-known regulators of meiotic HORMADs and required for their depletion from synapsed chromosomes. However, no direct interaction has been found between PCH2/TRIP13 and the presumptive HORMAD substrates in any organism other than in budding yeast. Thus, it remains largely elusive how the dynamics of ASY1 and other meiotic HORMADs are controlled. Here, we have identified COMET, the Arabidopsis homolog of human p31comet, which is known for its function in the spindle assembly checkpoint (SAC), as a central regulator of ASY1 dynamics in meiosis. We provide evidence that COMET controls ASY1 localization by serving as an adaptor for PCH2. Because ASY1 accumulates in the cytoplasm in early prophase and is persistently present on chromosomes in comet, we conclude that COMET is required for both the recruitment of ASY1 to the nucleus and the subsequent removal from the axis. The here-revealed function of COMET as an adaptor for PCH2 remarkably resembles the regulation of another HORMAD, Mad2, in the SAC in yeast and animals, revealing a conserved regulatory module of HORMA-domain-containing protein complexes.

Protein phosphatase 2A promotes stomatal development by stabilizing SPEECHLESS in Arabidopsis.

Stomatal guard cells control gas exchange that allows plant photosynthesis but limits water loss from plants to the environment. In Arabidopsis, stomatal development is mainly controlled by a signaling pathway comprising peptide ligands, membrane receptors, a mitogen-activated protein kinase (MAPK) cascade, and a set of transcription factors. The initiation of the stomatal lineage requires the activity of the bHLH transcription factor SPEECHLESS (SPCH) with its partners. Multiple kinases were found to regulate SPCH protein stability and function through phosphorylation, yet no antagonistic protein phosphatase activities have been identified. Here, we identify the conserved PP2A phosphatases as positive regulators of Arabidopsis stomatal development. We show that mutations in genes encoding PP2A subunits result in lowered stomatal production in Arabidopsis Genetic analyses place the PP2A function upstream of SPCH. Pharmacological treatments support a role for PP2A in promoting SPCH protein stability. We further find that SPCH directly binds to the PP2A-A subunits in vitro. In plants, nonphosphorylatable SPCH proteins are less affected by PP2A activity levels. Thus, our research suggests that PP2A may function to regulate the phosphorylation status of the master transcription factor SPCH in stomatal development.

Arabidopsis ITPK1 and ITPK2 Have an Evolutionarily Conserved Phytic Acid Kinase Activity.

Diphospho-myo-inositol polyphosphates, also termed inositol pyrophosphates, are molecular messengers containing at least one high-energy phosphoanhydride bond and regulate a wide range of cellular processes in eukaryotes. While inositol pyrophosphates InsP7 and InsP8 are present in different plant species, both the identity of enzymes responsible for InsP7 synthesis and the isomer identity of plant InsP7 remain unknown. This study demonstrates that Arabidopsis ITPK1 and ITPK2 catalyze the phosphorylation of phytic acid (InsP6) to the symmetric InsP7 isomer 5-InsP7 and that the InsP6 kinase activity of ITPK enzymes is evolutionarily conserved from humans to plants. We also show by 31P nuclear magnetic resonance that plant InsP7 is structurally identical to the in vitro InsP6 kinase products of ITPK1 and ITPK2. Our findings lay the biochemical and genetic basis for uncovering physiological processes regulated by 5-InsP7 in plants.

Lectin AtGAL1 interacts with high-mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate-starved Arabidopsis.

Among 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi-deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high-mannose AtPAP26-S2 glycoform with AtGAL1 (At1g78850), a Pi starvation-inducible (PSI), and Galanthus nivalis agglutinin-related (mannose-binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26-S2 to modify its enzymatic properties. Far-western immunodot blotting established that AtGAL1 readily associates with AtPAP26-S2 but not the low mannose AtPAP26-S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55-kDa AtGAL1 and AtPAP26-S2 polypeptides associate to form a 112-kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein-tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi-deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26-S2 but not AtPAP26-S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose-rich glycans of AtPAP26-S2 and consequent positive impact on AtPAP26-S2 activity and stability.

A glycoform of the secreted purple acid phosphatase AtPAP26 co-purifies with a mannose-binding lectin (AtGAL1) upregulated by phosphate-starved Arabidopsis.

The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn365 and Asn422 were modified in AtPAP26-S2 to form high-mannose glycans. A 55-kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr38 and Thr39 and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating posttranscriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.

Homologous pairing activities of Arabidopsis thaliana RAD51 and DMC1.

In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.

The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells.

The aim of this study was to determine the anticancer potential of Leonurus sibiricus extract derived from in vitro transgenic roots transformed by Agrobacetrium rhizogenes with AtPAP1 transcriptional factor, and that of transformed roots without construct, on grade IV human glioma cells and the U87MG cell line, and attempt to characterize the mechanism involved in this process. The anticancer effect induced by the tested extracts was associated with DNA damage, PARP cleavage/increased H2A.X histone levels and UHRF-1/DNMT1 down-regulation of mRNA levels. Additionally, we demonstrated differences in the content of compounds in the tested extracts by HPLC analysis with ATPAP1 construct and without. Both the tested extracts showed anticancer properties and the better results were observed for AtPAP1 with transcriptional factor root extract; this effect could be ascribed to the presence of higher condensed phenolic acids such as neochlorogenic acid, chlorogenic acids, ferulic acid, caffeic acid and p-coumaric acid. Further studies with AtPAP1 (with the transcriptional factor from Arabidopisi thaliana) root extract which showed better activities in combination with anticancer drugs are needed.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis.

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.

Ectodomain of plasmodesmata-localized protein 5 in Arabidopsis: expression, purification, crystallization and crystallographic analysis.

Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata of Arabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 from Arabidopsis was cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90 Šresolution and were indexed in space group P1, with unit-cell parameters a = 41.9, b = 48.1, c = 62.2 Å, α = 97.3, ß = 103.1, γ = 99.7°. Analysis of the crystal content indicated that there are two molecules in the asymmetric unit, with a Matthews coefficient of 2.51 Å3 Da-1 and a solvent content of 50.97%.

Recombinant ACHT1 from Arabidopsis thaliana: crystallization and X-ray crystallographic analysis.

Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in the Arabidopsis thaliana chloroplast. Recombinant ACHT1 protein was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Šresolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space group C2221, with unit-cell parameters a = 102.7, b = 100.6, c = 92.8 Å.

The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

Photoaffinity Labeling of Plant Receptor Kinases.

Defining the ligand-binding activity of receptors is important because the binding of ligands is the initial reaction in secreted ligand-dependent cell-to-cell communication. Photoaffinity labeling is one of the most efficient biochemical techniques for detecting direct ligand-receptor interactions. Here, we describe photoaffinity labeling to visualize the direct interaction between peptide ligands and their receptors by using photoactivatable and radioactive peptide ligand derivatives.

Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins.

Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using 14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins.

Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.

Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.

Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane.

To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 µg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.

Isolation of Endoplasmic Reticulum and Its Membrane.

The association of ribosomes with the rough endoplasmic reticulum (ER) is dependent on Mg2+. The ribosomes can be stripped from the ER by removal of Mg2+ from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when ER vesicles are analyzed by equilibrium density gradient centrifugation. Here, I describe the isolation of microsomes from Arabidopsis, followed by the use of the density shift approach in conjunction with equilibrium density gradient centrifugation as a means to diagnose whether a protein is associated with the ER. The same approach can also be used as a means to enrich for ER membranes.