Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana

Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

Identification and subcellular localization of the soybean copper P1B-ATPase GmHMA8 transporter

We have identified a copper P(1B)-ATPase transporter in soybean (Glycine max), named as GmHMA8, homologue to cyanobacterial PacS and Arabidopsis thaliana AtHMA8 (PAA2) transporters. A novel specific polyclonal anti-GmHMA8 antibody raised against a synthetic peptide reacted with a protein of an apparent mass of around 180-200 kDa in chloroplast and thylakoid membrane preparations isolated from soybean cell suspensions. Immunoblot analysis with this antibody also showed a band with similar apparent molecular mass in chloroplasts from Lotus corniculatus. Immunofluorescence labelling with the anti-GmHMA8 antibody and double immunofluorescence labelling with anti-GmHMA8 and anti-RuBisCo antibodies revealed the localization of the GmHMA8 transporter within the chloroplast organelle. Furthermore, the precise ultrastructural distribution of GmHMA8 within the chloroplast subcompartments was demonstrated by using electron microscopy immunogold labelling. The GmHMA8 copper transporter from soybean was localized in the thylakoid membranes showing a heterogeneous distribution in small clusters.     

Enzymatic properties and subcellular localization of Arabidopsis beta-N-acetylhexosaminidases

Plant glycoproteins contain substantial amounts of paucimannosidic N-glycans lacking terminal GlcNAc residues at their nonreducing ends. It has been proposed that this is due to the action of beta-hexosaminidases during late stages of N-glycan processing or in the course of N-glycan turnover. We have now cloned the three putative beta-hexosaminidase sequences present in the Arabidopsis (Arabidopsis thaliana) genome. When heterologously expressed as soluble forms in Spodoptera frugiperda cells, the enzymes (termed HEXO1-3) could all hydrolyze the synthetic substrates p-nitrophenyl-2-acetamido-2-deoxy-beta-d-glucopyranoside, p-nitrophenyl-2-acetamido-2-deoxy-beta-d-galactopyranoside, 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-d-glucopyranoside, and 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-d-glucopyranoside, albeit to a varying extent. HEXO1 to HEXO3 were further able to degrade pyridylaminated chitotriose, whereas pyridylaminated chitobiose was only cleaved by HEXO1. With N-glycan substrates, HEXO1 displayed a much higher specific activity than HEXO2 and HEXO3. Nevertheless, all three enzymes were capable of removing terminal GlcNAc residues from the alpha1,3- and alpha1,6-mannosyl branches of biantennary N-glycans without any strict branch preference. Subcellular localization studies with HEXO-fluorescent protein fusions transiently expressed in Nicotiana benthamiana plants showed that HEXO1 is a vacuolar protein. In contrast, HEXO2 and HEXO3 are mainly located at the plasma membrane. These results indicate that HEXO1 participates in N-glycan trimming in the vacuole, whereas HEXO2 and/or HEXO3 could be responsible for the processing of N-glycans present on secretory glycoproteins.     

Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L.: distinct SHG reside in the cytosol and in the apoplast

Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (<10 kDa; SHG(S)) and high (>10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using 14CO2 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime.     

Identification and characterization of rain, a novel Ras-interacting protein with a unique subcellular localization

The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras.     

Movement and subcellular localization of a tobamovirus in Arabidopsis

Tobamoviruses represent a well-characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus–plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis . Here, we describe infection of Arabidopsis by a recently discovered crucifer-infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis , causing mild and fast-developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus–plant interaction.     

Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO(4)) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.     

Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase

Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.     

Glycerate kinase from spinach leaves: Partial purification, characterization and subcellular localization

Spinach ( Spinacia oleracea L. cv. Melody hybrid) leaf glycerate kinase (EC 2.7.1.31) was partially purified and characterized. The enzyme did not exhibit any absolute stereospecificity towards the two enantiomers of glycerate, but the affinity for the D-isomer was 15-fold greater. The enzyme exhibited a broad pH optimum of 7.5–9.0 and a requirement for divalent cation, satisfied by Mg₂₊. The reaction product was identified as 3-phosphoglyceric acid. The observed high glycerate kinase activity together with its strategic localization exclusively in the chloroplast stroma are considered adequate for an efficient coupling of photosynthetic and photorespiratory carbon pathways.     

Purification, characterization and subcellular localization of pig liver alpha-L-iduronidase

alpha-L-Iduronidase was purified about 100,000-fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A-Sepharose 4B, heparin-Sepharose 4B, Toyopearl HW-55, Sephadex G-100 and chelating Sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G-100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium dodecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70-kDa and 62-kDa components were confirmed as alpha-L-iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0-3.5 and 65 microM 4-methylumbelliferyl-alpha-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5-6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.     

Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization

Clathrin fromH-K-ATPase-rich membranes derived from the tubulovesicular compartmentof rabbit and hog gastric acid secretory (parietal) cells wascharacterized biochemically, and the subcellular localization ofmembrane-associated clathrin in parietal cells was characterizedby immunofluorescence, electron microscopy, and immunoelectronmicroscopy. Clathrin from H-K- ATPase-rich membranes was determinedto be comprised of conventional clathrin heavy chain and a predominanceof clathrin light chain A. Clathrin and adaptors could be induced topolymerize quantitatively in vitro, forming 120-nm-diameter basketlikestructures. In digitonin-permeabilized resting parietal cells, theintracellular distribution of immunofluorescently labeled clathrin wassuggestive of labeling of the tubulovesicular compartment. Clathrin wasalso unexpectedly localized to canalicular (apical) membranes, as were-adaptin and dynamin, suggesting that this membrane domain ofresting parietal cells is endocytotically active. At theultrastructural level, clathrin was immunolocalized to canalicularand tubulovesicular membranes. H-K-ATPase was immunolocalized tothe same membrane domains as clathrin but did not appear to be enrichedat the specific subdomains that were enriched in clathrin. Finally, inimmunofluorescently labeled primary cultures of parietal cells, incontrast to the H-K-ATPase, intracellular clathrin was found not totranslocate to the apical membrane on secretagogue stimulation. Takentogether, these biochemical and morphological data provide a frameworkfor characterizing the role of clathrin in the regulation of membranetrafficking from tubulovesicles and at the canalicular membrane inparietal cells.

     

Asparagine and boric Acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase

Our previous work demonstrated substantial accumulation of allantoate in leaf tissue of nodulated soybeans (Glycine max L. Merr., cv Williams) in response to nitrogen fertilization. Research was continued to determine the effect of nitrate and asparagine on ureide assimilation in soybean leaves. Stem infusion of asparagine into ureide-transporting soybeans resulted in a significant increase in allantoate concentration in leaf tissue. Accumulation of allantoate was also observed when asparagine was supplied in the presence of allopurinol, an inhibitor of xanthine dehydrogenase in the pathway of ureide biosynthesis. In vitro, asparagine was found to have an inhibitory effect on the activity of allantoate amidohydrolase, a Mn²⁺-dependent enzyme catalyzing allantoate breakdown in soybean leaves. The inhibition was partially overcome by supplemental Mn²⁺ in enzyme assays. Another inhibitor of allantoate amidohydrolase, boric acid, applied foliarly on field-grown nodulated soybeans, caused up to a 10-fold increase in allantoate content of leaf tissue. Accumulation of allantoate in response to boric acid was either eliminated or greatly reduced in plants presprayed with Mn²⁺. We conclude that elevated levels of allantoate in leaves of ureide-transporting soybeans fertilized with ammonium nitrate result from inhibition of allantoate degradation by asparagine and that Mn²⁺ is a critical factor in this inhibition. Furthermore, our studies with asparagine and boric acid indicate that availability of Mn²⁺ has a direct effect on ureide catabolism in soybean.     

Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The Vmax/Km values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana.     

Diacylglycerol kinase from suspension cultured plant cells : characterization and subcellular localization

Diacylglycerol kinase (adenosine 5′-triphosphate:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5′-triphosphate and guanosine 5′-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane.     

Molecular characterization, tissue distribution, subcellular localization and actin-sequestering function of a thymosin protein from silkworm

We identified a novel gene encoding a Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development.     

Identification of the subcellular localization of mycobacterial proteins using localization motifs

Mycobacterium, the most common disease-causing genus, infects billions of people and is notoriously difficult to treat. Understanding the subcellular localization of mycobacterial proteins can provide essential clues for protein function and drug discovery. In this article, we present a novel approach that focuses on local sequence information to identify localization motifs that are generated by a merging algorithm and are selected based on a binomially distributed model. These localization motifs are employed as features for identifying the subcellular localization of mycobacterial proteins. Our approach provides more accurate results than previous methods and was tested on an independent dataset recently obtained from an experimental study to provide a first and reasonably accurate prediction of subcellular localization. Our approach can also be used for large-scale prediction of new protein entries in the UniportKB database and of protein sequences obtained experimentally. In addition, our approach identified many local motifs involved with the subcellular localization that also interact with the environment. Thus, our method may have widespread applications both in the study of the functions of mycobacterial proteins and in the search for a potential vaccine target for designing drugs.