No abstract

Rice seeds, a rich reserve of starch and protein, are a major food source in many countries. Unlike the seeds of other plants, which typically accumulate one major type of storage protein, rice seeds use two major classes, prolamines and globulin-like glutelins. Both storage proteins are synthesized on the endoplasmic reticulum (ER) and translocated to the ER lumen, but are then sorted into separate intracellular compartments. Prolamines are retained in the ER lumen as protein bodies whereas glutelins are transported and stored in protein storage vacuoles. Mechanisms responsible for the retention of prolamines within the ER lumen and their assembly into intracisternal inclusion granules are unknown, but the involvement of RNA localization has been suggested. Here we show that the storage protein RNAs are localized to distinct ER membranes and that prolamine RNAs are targeted to the prolamine protein bodies by a mechanism based on RNA signal(s), a process that also requires a translation initiation codon. Our results indicate that the ER may be composed of subdomains that specialize in the synthesis of proteins directed to different compartments of the plant endomembrane system.

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through proteinprotein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.In prokaryotic organisms, the PII protein is recognized as the key mediator of energy, carbon, and nitrogen interactions and is referred to as the central processing unit of carbon:nitrogen metabolism (1-4). Escherichia coli PII is a 112-amino acid protein that as a homotrimer senses the cellular status of both ATP and the carbon skeleton 2-oxoglutarate (2KG) 3 via allosteric means. Nitrogen status is assessed through glutamine levels by covalent modification (uridylylation) of PII. This metabolic information is signaled to other proteins by proteinprotein interaction and produces an appropriate response that alters gene expression and the activity of glutamine synthetase (3, 5). In terms of metabolic sensing, cyanobacterial PII plays a similar role, but in this case, covalent modification is by phosphorylation (6). To date, the processes known to be regulated by PII in cyanobacteria are: ammoniumdependent nitrate/nitrite uptake (7), high affinity bicarbonate transport (8), regulation of the global transcriptional activation by NtcA (9, 10), and arginine biosynthesis (11).In eukaryotes, PII has only been identified in plants and red algae (12), and its sequence is highly conserved when compared with prokaryotic PIIs, with Arabidopsis thaliana PII being 50 and 55% identical to E. coli and Synechococcus elongatus PII, respectively. Plant PII proteins have a conserved N-terminal extension that functions as a chloroplast transit peptide, which is consistent with biochemical data indicating that PII resides in this compartment. We have previously shown that the plant PII protein is not regulated by phosphorylation (13). Like the bacterial...

Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells. Molecular & Cellular Proteomics 3:970 -983, 2004.The cytoskeleton is the single most important structure that contributes to the highly ordered organization of the eukaryotic cell. It provides a framework for cell division and the trafficking of organelles and macromolecules, and also serves to regulate important cellular processes such as signaling, translation, and metabolism. The cytoskeleton plays a key role in a number of plant-specific processes, such as assisting in the formation of the cell plate, regulating cell-to-cell movement, and influencing the direction of cell elongation (1). A role for the microtubule (MT) 1 component of the cytoskeleton in many of these processes has been demonstrated, and a number of MT-binding proteins that are responsible for regulating these events have been identified.Plant MTs are assembled into four distinct arrays during the cell cycle (2). Three of these arrays-the interphase cortical array, the pre-prophase band, and the phragmoplast-have no counterpart in animal cells. The cortical MT array has been linked to the regulation of cellulose microfibril deposition and, hence, a role in cell expansion, while the pre-prophase band and the phragmoplast have important roles in the positioning and synthesis of the new cell plate in dividing cells. The fourth array, the spindle, has an evolutionarily conserved role in the segregation of chromosomes during cell division. The organization and dynamics of MTs in these arrays depend on the activity of various MT-associated proteins (MAPs). Several plant MAPs have been identified, including the 65-kDa MAPs, MAP 190, and MOR1 (3). These proteins are important in cross-bridging MTs, linking MTs with actin filamen...

BackgroundPuf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants.ResultsThe Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus.ConclusionsThe Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.

An important principle of the functional organization of plant cells is the targeting of proteins to specific subcellular locations. The physical location of proteins within the apoplasm/ rhizosphere at the root-soil interface positions them to play a strategic role in plant response to biotic and abiotic stress. We previously demonstrated that roots of Triticum aestivum and Brassica napus exude a large suite of proteins to the apoplasm/rhizosphere [Basu et al. (1994) Plant Physiol 106:151-158; Basu et al. (1999) Physiol Plant 106:53-61]. This study is a first step to identify low abundance extracytosolic proteins from Arabidopsis thaliana and Brassica napus roots using recent advances in the field of proteomics. A total of 16 extracytosolic proteins were identified from B. napus using two-dimensional gel electrophoresis, tandem mass spectrometry (LC-MS/MS) and de novo sequencing. Another high-throughput proteomics approach, Multidimensional Protein Identification Technology (Mud PIT) was used to identify 52 extracytosolic proteins from A. thaliana. Signal peptide cleavage sites, the presence/absence of transmembrane domains and GPI modification were determined for these proteins. Functional classification grouped the extracellular proteins into different families including glycoside hydrolases, trypsin/ protease inhibitors, plastocyanin-like domains, copper-zinc superoxide dismutases, gamma-thioinins, thaumatins, ubiquitins, protease inhibitor/ seed storage/lipid transfer proteins, transcription factors, class III peroxidase, and plant basic secretory proteins (BSP). We have also developed an on-line, Extracytosolic Plant Proteins Database (EPPdb, http://eppdb.biology.ualberta.ca) to provide information about these extracytosolic proteins.

In order to improve our understanding of the host cell-parasite interactions in giardiasis, this study assessed the effects of Giardia lamblia on epithelial permeability and tight junctional ZO-1, determined whether epidermal growth factor (EGF) may affect Giardia-induced epithelial injury, and evaluated if EGF modulates epithelial colonization by live G. lamblia trophozoites. Permeability was assessed in assays of trans-epithelial fluxes of FITC-dextran, and ZO-1 integrity was characterized by confocal laser immunofluorescence microscopy in confluent epithelial cell monolayers. G. lamblia significantly increased paracellular permeability and disrupted tight-junctional ZO-1 of a novel non-transformed human small intestinal epithelial cell line (SCBN). Pre-treatment with EGF prevented the development of these abnormalities and significantly inhibited attachment of live trophozoites to the enterocytes, independently of a direct microbiocidal action. These findings demonstrate that G. lamblia may cause intestinal pathophysiology by disrupting tight junctional ZO-1 and increasing epithelial permeability. Apical administration of EGF prevents these abnormalities, and reduces epithelial colonization by the live parasites.

SummaryDual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.