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T Leustek

Publications and source records attributed to T Leustek.

At least 37 records · Page 2Linked to original sources
PubMed1997

Regulation of sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana.

Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants. Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation. In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and serine acetyltransferase (SAT1) were inducible by sulfate starvation in A. thaliana. The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.

Amino Acid Sequence↗
PubMed1997

A new route for synthesis of dimethylsulphoniopropionate in marine algae.

The 3-dimethylsulphoniopropionate (DMSP) produced by marine algae is the main biogenic precursor of atmospheric dimethylsulphide (DMS). This biogenic DMS, formed by bacterial and algal degradation of DMSP, contributes about 1.5 x 10(13) g of sulphur to the atmosphere annually, and plays a major part in the global sulphur cycle, in cloud formation and potentially in climate regulation. Although DMSP biosynthesis has been partially elucidated in a higher plant, nothing is known about how algae make DMSP except that the whole molecule is derived from methionine. Here we use in vivo isotope labelling to demonstrate that DMSP synthesis in the green macroalga Enteromorpha intestinalis proceeds by a route entirely distinct from that in higher plants. From methionine, the steps are transamination, reduction and S-methylation to give the novel sulphonium compound 4-dimethylsulphonio-2-hydroxybutyrate (DMSHB), which is oxidatively decarboxylated to DMSP. The key intermediate DMSHB was also identified in three diverse phytoplankton species, indicating that the same pathway operates in other algal classes that are important sources of DMS. The fact that a transamination initiates this pathway could help explain how algal DMSP (and thereby DMS) production is enhanced by nitrogen deficiency.

Chlorophyta↗
PubMed1997

Siroheme biosynthesis in higher plants. Analysis of an S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana.

Siroheme, the prosthetic group for both nitrite and sulfite reductases, is a methylated, iron-containing modified tetrapyrrole. Here we report the first molecular characterization of the branch point enzyme in higher plants, which directs intermediates toward siroheme synthesis. A cDNA was cloned from Arabidopsis thaliana (UPM1) that functionally complements an Escherichia coli cysG mutant, a strain that is unable to catalyze the conversion of uroporphyrinogen III (Uro'gen-III) to siroheme. UPM1 is 1484 base pairs and encodes a 369-amino acid, 39.9-kDa protein. The UPM1 product contains two regions that are identical to consensus sequences found in bacterial Uro'gen-III and precorrin methyltransferases. Recombinant UPM1 protein was found to catalyze S-adenosyl-L-methionine-dependent transmethylation by UPM1 in a multistep process involving the formation of a covalently linked complex with S-adenosyl-L-methionine. The UPM1 product has a sequence at the amino terminus that resembles a transit peptide for localization to mitochondria or plastids. The protein produced by in vitro expression is able to enter isolated intact chloroplasts but not mitochondria. Genomic blot analysis showed that UPM1 is encoded in the A. thaliana genome. The genomic DNA corresponding to UPM1 was cloned and sequenced and found to contain at least five introns.

Amino Acid Sequence↗
PubMed1996

Sulfate reduction in higher plants: molecular evidence for a novel 5'-adenylylsulfate reductase.

Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5'-phosphosulfate (APS) (5'-adenylysulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. Further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5'-adenosinemonophosphate (5'-AMP). 2'-AMP, or 3'-phosphoadenosine-5'-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.

Amino Acid Sequence↗
PubMed1996

Heat shock enhances thermotolerance of infective juvenile insect-parasitic nematodes Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae).

Insect-parasitic nematodes possess many of the attributes of ideal biological control agents, but intolerance to extreme temperatures can restrict their use. We examined whether heat-shock treatments could improve nematode survival and infectivity at temperatures that normally inhibit their activity (35 and 40 degrees C). Nematodes exposed to a sub-lethal temperature (35 degrees C) for 3 h with a latency period of 1-2 h at 25 degrees C killed insects at 35 and 40 degrees C. Correlative evidence was obtained between increased thermotolerance and the synthesis of 70-kDa heat-shock proteins (hsps). These results provide the first evidence of hsp synthesis in the development of thermotolerance and biological activity in the non-feeding, developmentally arrested, infective juvenile nematodes.

Animals↗
PubMed1996

Cloning and analysis of the gene for cystathionine gamma-synthase from Arabidopsis thaliana.

A cDNA clone, CGS1, encoding cystathionine gamma-synthase (CGS) from Arabidopsis thaliana was selected by complementation of CGS mutant strain of Escherichia coli (metB). Cells expressing CGS1 can grow on medium lacking Met and contain CGS enzyme activity. Genomic DNA blot analysis of A. thaliana revealed that there is a single gene homologous with CGS1. A genomic fragment carrying CGS1 was cloned and sequenced. Through combined analysis of the cDNA and genomic clone it was determined that the CGS1 coding sequence is 1692 bp, encodes a 563 amino acid, 60 kDa protein, and is interrupted by ten introns. A transcriptional initiation site was detected 260 bp 5' of the initiator codon. The predicted amino acid sequence of CGS1 contains a consensus pyridoxal phosphate-binding site and is similar to MetB of E. coli, with which it is 35 percent identical. The CGS1 product has a sequence at the amino terminus that resembles a transit peptide for localization to plastids. At least 160 amino acids from the amino terminus of the CGS1 enzyme are not essential for enzymatic activity.

Amino Acid Sequence↗
PubMed1995

A multifunctional Urechis caupo protein, PAPS synthetase, has both ATP sulfurylase and APS kinase activities.

The synthesis of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from inorganic sulfate and ATP requires two enzymes, ATP sulfurylase (SUL) and adenosine-5'-phosphosulfate kinase (KIN). In bacteria, fungi, yeast and plants, the two enzymes are present on separate polypeptide chains. We have identified the first animal gene coding for these enzymes. In the marine worm, Urechis caupo (Uc), both SUL and KIN are present on a single polypeptide chain. This protein, which we call PAPS synthetase (PAPSS), is able to complement yeast mutants lacking either enzyme. The Uc PAPSS mRNA is present in oocytes, but is not translated until after fertilization. At least three adult tissues, gut, ceolomocytes and body wall, also contain the mRNA, but at lower concentrations than are found in embryos. Partial sequences of a similar gene from Caenorhabditis elegans (Ce) were detected in a search of the GenBank expressed sequence tag database. Comparison of these Uc and Ce PAPSS sequences with the sequences of cloned genes from non-animal organisms strongly suggests that the animal genes evolved through the fusion of the SUL- and KIN-encoding genes from lower organisms.

Amino Acid Sequence↗
PubMed1995

Adenosine-5'-triphosphate-sulfurylase from Arabidopsis thaliana and Escherichia coli are functionally equivalent but structurally and kinetically divergent: nucleotide sequence of two adenosine-5'-triphosphate-sulfurylase cDNAs from Arabidopsis thaliana and analysis of a recombinant enzyme.

ATP-sulfurylase, the first enzyme of sulfate assimilation, catalyzes the formation of adenosine-5'-phosphosulfate from ATP and sulfate. Here we report that the higher plant, Arabidopsis thaliana, contains a three-member, expressed gene family encoding plastid localized forms of ATP sulfurylase. Three cDNAs from A. thaliana, designated APS1, APS2, and APS3, were isolated by their ability to functionally complement a met3 (ATP sulfurylase) mutant strain of Saccharomyces cerevisiae (yeast). The nucleotide sequence of APS1 was reported previously (1). APS2 and APS3, reported here, have 476- and 465-codon open-reading frames encoding 53.6- and 52.0-kDa polypeptides, respectively. The translation products of both clones are highly homologous to APS1 (66 and 86% identity, respectively) over their entire lengths, including amino terminal sequences resembling transit peptides for plastid localization. Both clones are less homologous to MET3 (25 and 30% identity, respectively). Genomic blot analysis of A. thaliana revealed only three genes with homology to the APS cDNAs and RNA blot analysis showed that APS1 is the most highly expressed member of this gene family. The APS polypeptides share homology with ATP-sulfurylases from fungi, a marine worm and a chemoautotrophic bacterium, but, not from Escherichia coli or Rhizobium meliloti. Analysis of recombinant APS3 showed that the protein is structurally and kinetically similar to fungal ATP-sulfurylase, but very different from the E. coli enzyme. The APS3 polypeptide is a homotetramer with specific activities (mumol primary product x mg protein-1 at pH 8.0, 25 degrees C) for 2.9 for APS synthesis, 30.1 for molybdolysis, and 48.7 for ATP synthesis. Despite the sequence, structural, and kinetic differences between higher plant and E. coli ATP-sulfurylases, APS2 and APS3 are able to functionally complement E. coli cysD and cysN (ATP-sulfurylase) mutant strains.

Amino Acid Sequence↗
PubMed1995

Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation.

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.

Amino Acid Sequence↗
PubMed1995

Serine acetyltransferase from Arabidopsis thaliana can functionally complement the cysteine requirement of a cysE mutant strain of Escherichia coli.

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway of sulfate assimilating organisms, catalyzes the formation of O-acetylserine, the immediate precursor of L-cysteine. In higher plants, it is thought that sulfur assimilation occurs primarily in leaf chloroplasts; however, serine acetyltransferase is not localized exclusively in this tissue and organelle. At least three genes for serine acetyltransferase have been identified in the higher plant Arabidopsis thaliana. Reported here is a cDNA corresponding to one of these genes, SAT1, a 1,079 bp clone with an open reading frame predicted to encode a 34-kDa protein that is able to functionally complement a serine acetyltransferase mutant strain of Escherichia coli. The predicted amino acid sequence of SAT1 shows significant homology with bacterial serine acetyltransferases. SAT1, expressed as a recombinant protein, shows serine acetyltransferase enzyme activity and cross-reacts with an antibody against the homologous E. coli enzyme. The first 40 amino acids of the SAT1 polypeptide resembles a plastid transit peptide, but the polypeptide is probably not plastid localized. Genomic DNA blot analysis of A. thaliana showed that SAT1 is a single copy gene and RNA blot analysis revealed that SAT1 is expressed in both leaves and roots.

Acetyltransferases↗
PubMed1994

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).

Amino Acid Sequence↗
PubMed1993

Characteristics of an Hsp70 homolog localized in higher plant chloroplasts that is similar to DnaK, the Hsp70 of prokaryotes.

Members of the 70-kD heat-shock protein (Hsp70) family are important cellular factors that are thought to mediate protein folding and assembly. A chloroplast-localized Hsp70 homolog (Chsp70) was recently identified based on its similarity to DnaK, the Hsp70 homolog of Escherichia coli (D. Amir-Shapira, T. Leustek, B. Dalie, H. Weissbach, N. Brot [1990] Proc Natl Acad Sci USA 87: 1749-1752). To learn more about the function of Chsp70, we purified the protein from Spinacia oleracea chloroplasts by ATP-agarose affinity chromatography. A single, 75,000-D protein was isolated which becomes phosphorylated on a threonine residue when incubated with [gamma-32P]ATP and 10 mM Ca2+, a property similar to DnaK. Chloroplast fractionation and immunoblot analysis showed that Chsp70 is a soluble stromal protein. Chsp70-specific antiserum was used to clone a partial cDNA that shows greater homology with Hsp70 from prokaryotes than with cytoplasmic Hsp70 from eukaryotes. The antiserum and cDNA were used to study Chsp70 expression. Following heat shock of spinach seedlings at 37 degrees C, Chsp70 synthesis increase 12-fold, the level of Chsp70 mRNA increases 5-fold, and the level of Chsp70 protein increases less than 2-fold. Chsp70 is constitutively expressed in all spinach tissues, indicating that it is likely to be localized in all plastid types. The highest levels occur in seeds, leaves, florets, and seedlings grown in the light. Lower levels occur in roots, stems, and etiolated seedlings.

Bacterial Proteins↗
PubMed1992

Autophosphorylation of 70 kDa heat shock proteins.

Several members of the 70 kDa heat shock protein group are known to be phosphorylated in vivo and have recently been found to undergo a Ca(2+)-stimulated autophosphorylation. The characteristics of the autophosphorylation reaction with Escherichia coli DnaK the mitochondrial and chloroplast homologs, and the endoplasmic reticulum Bip/Grp78 are discussed. Some common features are a requirement for Ca2+, inhibition by Mg2+ and phosphorylation solely on a threonine residue. Although the role of autophosphorylation of these proteins is not clear, it is known that the level of phosphorylation of some Hsp70 proteins in vivo is responsive to stress and other cellular conditions.

Calcium↗
PubMed1991

Calcium-dependent autophosphorylation of the glucose-regulated protein, Grp78.

The characteristics of phosphorylation of the 78-kDa glucose-regulated protein (Grp78), also known as the immunoglobulin heavy chain binding protein, were studied in vitro and in vivo. The purified protein from either calf liver or bovine kidney cells (MDBK) could be phosphorylated in vitro with [gamma-32P]ATP, in a reaction that is stimulated by Ca2+ and inhibited by the Ca(2+)-chelator ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). In the presence of EGTA, excess Ca2+ increased the rate of phosphorylation about 18-fold. Based on EGTA/Ca2+ titrations, the optimal Ca2+ concentration for phosphorylation was estimated to be 10-50 microM. Other divalent cations such as Mg2+, Mn2+, and Zn2+ were found to be inhibitory as was the Ca2+ antagonist lanthanum (La3+). The in vivo phosphorylation of Grp78 was studied in MDBK cells labeled with 32Pi. In the presence of inducers of Grp78 synthesis, such as ionomycin, tunicamycin, or 2-deoxyglucose, there was a large increase in the level of Grp78 in the cells but a decrease in the amount of phosphorylated protein. Two-dimensional gel analysis of Grp78 purified from bovine liver and MDBK cells identified at least four isoforms. After in vivo and in vitro phosphorylation of Grp78 all the acidic isoforms contained radioactivity but not the most basic isoform. Phosphoamino acid analysis of Grp78 showed that serine and threonine were phosphorylated in vivo and only threonine was phosphorylated in vitro.

Adenosine Triphosphate↗
PubMed1990

Hsp70 proteins, similar to Escherichia coli DnaK, in chloroplasts and mitochondria of Euglena gracilis.

The heat-shock response of Euglena gracilis was studied by pulse-labeling cells with [35S]sulfate at both the normal growth temperature (21 degrees C) and an elevated temperature (36 degrees C). Analysis of the labeled proteins by polyacrylamide gel electrophoresis indicated that the rate of synthesis of at least 3 major and 15 minor polypeptides increased in cells grown at the higher temperature. Three of the proteins appear to be immunologically related to the ubiquitous approximately 70-kDa heat-shock protein (Hsp70) family. One protein of 68 kDa was found in the cytoplasm (P68cyt) and was the major heat-shock protein in Euglena gracilis. Two other proteins, 68 and 70 kDa, were localized in mitochondria (P68mit) and chloroplasts (P70chl), respectively, and they crossreacted with a polyclonal antibody raised against the Escherichia coli heat-shock protein DnaK. Like DnaK, P68mit and P70chl could be phosphorylated in vitro with [gamma-32P]ATP in a reaction that was stimulated by Ca2+. A protein with characteristics similar to those of P70chl was also found in chloroplasts isolated from maize and spinach.

Animals↗
PubMed1990

Selection and physiology of cell cultures of Douglas-fir grown under conditions of water stress.

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) cell cultures sampled 3, 6, or 9 days after subculture in nutrient medium were able to survive subsequent subculture in a medium containing 15% polyethylene glycol (PEG) (M(r) 6000-8000) (-1.21 MPa), whereas cell sampled 12 or 16 days after subculture in nutrient medium became senescent when transferred to a medium containing 15% PEG. Cells sampled after subculture for 3, 6, or 9 days in nutrient medium had lower fresh weight/dry weight ratios, lower osmotic potentials, smaller cell diameters, and higher turgor pressures than cells sampled after 12 or 16 days subculture in nutrient medium. Cells surviving subculture to a medium containing 15% PEG did not increase in dry weight for 5 weeks even though the medium was exchanged every 7 days. After 5 weeks, however, dry weight growth resumed and reached 75% of the level attained by control cells grown on PEG-free medium. Long-term growth on a medium containing 15% PEG (PEG-selected cells) could only be sustained if the medium was supplemented with 30 mM glutamine. The PEG-selected cells grew in small clusters, were isodiametric, and had chlorophyll contents 50% higher than unselected cells. The PEG-selected cells also showed lowered cellular osmotic potentials, presumably due to osmoregulation. Turgor pressures of PEG-selected cells were greater than or equal to those of unselected cells.

Journal Article↗