Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

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.

Isolation of Plastoglobules for Lipid Analyses.

Plastoglobules are plastid compartments designed for the storage of neutral lipids. They share physical and structural characteristics with cytosolic lipid droplets. Hence, special care must be taken to avoid contamination by cytosolic lipid droplets during plastoglobule purification. We describe the isolation of pure plastoglobules from Arabidopsis thaliana leaves, and the methods we use to determine their lipid composition. After preparation of a crude chloroplast fraction, plastoglobules are isolated from plastid membranes by two steps of ultracentrifugation on discontinuous sucrose gradients. For lipid analyses, total lipids are then extracted by a standard chloroform-methanol protocol, and polar lipids are separated from neutral lipids by liquid-liquid extraction. While polar lipid classes are subsequently separated by thin-layer chromatography (TLC) with the classical Vitiello solvent mix, a double TLC development has to be performed for neutral lipids, to separate phytyl and steryl esters. Lipids are quantified by gas chromatography after conversion of the fatty acids into methyl esters.

Isolation of Mitochondria for Lipid Analysis.

Diverse classes of lipids are found in cell membranes, the major ones being glycerolipids, sphingolipids, and sterols. In eukaryotic cells, each organelle has a specific lipid composition, which defines its identity and regulates its biogenesis and function. For example, glycerolipids are present in all membranes, whereas sphingolipids and sterols are mostly enriched in the plasma membrane. In addition to phosphoglycerolipids, plants also contain galactoglycerolipids, a family of glycerolipids present mainly in chloroplasts and playing an important role in photosynthesis. During phosphate starvation, galactoglycerolipids are also found in large amounts in other organelles, illustrating the dynamic nature of membrane lipid composition. Thus, it is important to determine the lipid composition of each organelle, as analyses performed on total cells do not represent the specific changes occurring at the organelle level. This task requires the optimization of standard protocols to isolate organelles with high yield and low contamination by other cellular fractions. In this chapter, we describe a protocol to isolate mitochondria from Arabidopsis thaliana cell cultures to perform lipidomic analysis.

Collection and Analysis of Phloem Lipids.

The plant phloem is a long-distance conduit for the transport of assimilates but also of mobile developmental and stress signals. These signals can be sugars, metabolites, amino acids, peptides, proteins, microRNA, or mRNA. Yet small lipophilic molecules such as oxylipins and, more recently, phospholipids have emerged as possible long-distance signals as well. Analysis of phloem (phospho)lipids, however, requires enrichment, purification, and sensitive analysis. This chapter describes the EDTA-facilitated approach of phloem exudate collection, phase partitioning against chloroform-methanol for lipid separation and enrichment, and analysis/identification of phloem lipids using LC-MS with multiplexed collision induced dissociation (CID).

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.

Phytochrome B interacts with SWC6 and ARP6 to regulate H2A.Z deposition and photomorphogensis in Arabidopsis.

Light serves as a crucial environmental cue which modulates plant growth and development, and which is controlled by multiple photoreceptors including the primary red light photoreceptor, phytochrome B (phyB). The signaling mechanism of phyB involves direct interactions with a group of basic helix-loop-helix (bHLH) transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and the negative regulators of photomorphogenesis, COP1 and SPAs. H2A.Z is an evolutionarily conserved H2A variant which plays essential roles in transcriptional regulation. The replacement of H2A with H2A.Z is catalyzed by the SWR1 complex. Here, we show that the Pfr form of phyB physically interacts with the SWR1 complex subunits SWC6 and ARP6. phyB and ARP6 co-regulate numerous genes in the same direction, some of which are associated with auxin biosynthesis and response including YUC9, which encodes a rate-limiting enzyme in the tryptophan-dependent auxin biosynthesis pathway. Moreover, phyB and HY5/HYH act to inhibit hypocotyl elongation partially through repression of auxin biosynthesis. Based on our findings and previous studies, we propose that phyB promotes H2A.Z deposition at YUC9 to inhibit its expression through direct phyB-SWC6/ARP6 interactions, leading to repression of auxin biosynthesis, and thus inhibition of hypocotyl elongation in red light.

Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV.

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

Isolation of UVR8 Protein Complexes.

The fundamental mechanism of light regulated plant development involves photoreceptors and their interacting proteins which act as light signaling intermediate factors. In Arabidopsis thaliana, UV RESISTANCE LOCUS 8 (UVR8) is responsible for the perception and the initiation of UV-B light signal. To data, only a few proteins have been revealed as the components of UVR8 protein complexes, limiting our understanding of the molecular mechanisms by which UV-B light input is interpreted to orchestrate numerous physiological outputs in plants. Therefore, it is necessary to isolate and identify the components of UVR8 protein complexes at a global level, in order to uncover novel UV-B light signaling factors and pathways. In this chapter, we provide a protocol for the isolation of UVR8 protein complexes. Basically, co-immunoprecipitation (co-IP) assay is employed to enrich UVR8 and its associating proteins in vivo. This method can be used coupling with specific treatments and is compatible with successive biochemical analysis.

Express Arabidopsis Cryptochrome in Sf9 Insect Cells Using the Baculovirus Expression System.

The Bac-to-Bac® Baculovirus Expression System provides a rapid and efficient method to generate recombinant cryptochrome (CRY) proteins with chromophore flavin (FAD), which showed blue light response in vitro.

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.

Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

Identification of ABA Receptor Agonists Using a Multiplexed High-Throughput Chemical Screening.

Small molecules that can activate abscisic acid (ABA) receptors represent valuable probes to study ABA perception and signaling. Additionally, these compounds have the potential to be used in the field to counteract the negative effect of drought stress on plant productivity. The PYR/PYL ABA receptors, in their ligand-bound conformation, inactivate protein phosphatases 2C (PP2Cs), triggering physiological responses that are essential for plant adaptation to environmental stresses, including drought. Based on this ligand-induced PP2C inactivation mechanism, we have developed an in vitro assay for the identification of ABA-receptor agonists by high-throughput screening of chemical libraries. The assay allows simultaneous use of different ABA receptors, increasing the chances to find new agonists and eliminates the need for parallel screening. In this chapter, we describe detailed procedures for the identification of ABA agonists using this multiplexed assay in a medium- (96-well plates) or a high-throughput (384-well plates) setup.

Label-Free Target Identification and Confirmation Using Thermal Stability Shift Assays.

Target identification presents one of the biggest challenges to chemical genomic approaches. In recent years, several methods have been applied for target identification and validation in plant cells. Here, we describe a label-free method based on the thermodynamic stabilization of a protein by interaction with a small-molecule ligand. With increasing temperature, proteins undergo thermal denaturation resulting in irreversible aggregation and precipitation. The binding of a small molecule to its target can enhance protein stability resulting in an increased temperature of aggregation (Tagg). This distinct increase in the temperature of aggregation known as a thermal shift can identify a compound-target protein interaction in high-throughput assays or, validate a predicted interaction.

Microscale Thermophoresis (MST) to Detect the Interaction Between Purified Protein and Small Molecule.

Microscale thermophoresis (MST) is a biophysical assay to quantify the interaction between molecules, such as proteins and small molecules. In recent years, the MST assay has been used to detect protein-protein and protein-drug interactions. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent molecules in response to a temperature gradient. In practice, the fluorescent molecule is mixed with different concentrations of the nonfluorescent ligand, and the mixture of molecules in solution is loaded to capillaries. A temperature gradient is applied to samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded. The effect of different concentrations of the nonfluorescent ligand on the movement of the fluorescent molecule is quantified to test for the interaction between molecules. If the fluorescent molecule interacts with the ligand, the molecular properties of the molecules, such as charge, size, and hydration shell, will influence the molecular motility. MST has the advantages of being quantitative and robust. In this chapter, we will use Endosidin2 and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1), as an example to show the procedure of using MST to test the interaction between a GFP-tagged protein and a small molecule.

AtTrxh3, a Thioredoxin, Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana.

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.

The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation.

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]2+ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]2+ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]2+ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]2+ clusters, via two-electron reductive coupling of two [2Fe-2S]2+ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.

Expression, purification, and phylogenetic analysis of MDIS1-INTERACTING RECEPTOR-LIKE KINASE1 (MIK1).

An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC-MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.

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.

Isolation and Glycomic Analysis of Trans-Golgi Network Vesicles in Plants.

The dynamic endomembrane system facilitates sorting and transport of diverse cargo. Therefore, it is crucial for plant growth and development. Vesicle proteomic studies have made substantial progress in recent years. In contrast, much less is known about the identity of vesicle compartments that mediate the transport of polysaccharides to and from the plasma membrane and the types of sugars they selectively transport. In this chapter, we provide a detailed description of the protocol used for the elucidation of the SYP61 vesicle population glycome. Our methodology can be easily adapted to perform glycomic studies of a broad variety of plant cell vesicle populations defined via subcellular markers or different treatments.