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Biomedical subjects

A Wiemken

Publications and source records attributed to A Wiemken.

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

Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock.

Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while TsI1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, TsI1, and Tps3, a protein with high homology to TsI1, revealed that both TsI1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that TsI1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/phosphatase complex in exponentially growing, heat-shocked cells.

Bacterial Proteins↗
PubMed1997

Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe.

Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition. This is accompanied by induction of heat-shock protein (hsp) synthesis and rapid accumulation of trehalose. Genetic approaches to determine the specific role of trehalose in heat-induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1, the gene encoding trehalose-6-phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose-containing media. Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects. We show that tps1 mutants have a serious defect in heat shock-induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40-42.5 degrees C), which, in wild-type cells, prevent hsp but not trehalose synthesis. In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e. 35 degrees C). In addition, pka1 mutants deficient in cAMP-dependent protein kinase were examined. Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance. It was found that pka1 mutant cells, contrary to wild-type cells, accumulated large amounts of trehalose, even during a 50 degrees C treatment. pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock.

Adaptation, Physiological↗
PubMed1997

Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase.

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.

Allium↗
PubMed1996

Inulin synthesis by a combination of purified fructosyltransferases from tubers of Helianthus tuberosus.

Sucrose-sucrose 1-fructosyltransferase (1-SST) was purified 100-fold from tubers of Helianthus tuberosus L. The purified enzyme was essentially devoid of invertase activity and could be separated by isoelectric focusing into five isoforms which all were composed of two subunits (59 and 26 kDa). Fructan-fructan 1-fructosyltransferase (1-FFT) was purified from the same source [M. Lüscher et al. (1993) New Phytologist 123, 437-442). When incubated individually with sucrose, 1-FFT was inactive while 1-SST formed isokestose (trimer) and, upon prolonged incubation, some nystose (tetramer). When a combination of the two enzymes was incubated with sucrose, a series of oligofructosides with a degree of polymerization of up to 20 was formed. Amino acid sequences of tryptic peptide fragments from both 1-SST and 1-FFT indicate that these enzymes are highly homologous with plant invertases.

Amino Acid Sequence↗
PubMed1996

Proteolytic processing of class IV chitinase in the compatible interaction of bean roots with Fusarium solani.

Three chitinase isoenzymes, PvChiE, PvChiF, and PvChiG (molecular masses 29, 28, 27 kD, respectively), were purified from bean (Phaseolus vulgaris L. cv Saxa) roots infected with the fungal pathogen Fusarium solani f. sp. phaseoli, and their amino acid sequence was partially determined. All sequences from all three isoenzymes exactly matched deduced amino acid sequences of the bean class IV chitinase PvChi4, formerly called PR4. The N terminus of PvChif mapped to the hinge region, and the N terminus of PvChiG mapped to the catalytic domain of PvChi4. The N terminus of PvChiE was blocked. The appearance of PvChiE, PvChiF, and PvChiG correlated with an increase in protease activity in infected roots, and they could be generated in vitro by mixing extracts with high protease activity with extracts containing high amounts of PvChi4. Extracts from infected roots prepared in the presence of protease inhibitors also contained the processed forms of PvChi4, indicating that processing occurred in planta and not as an artifact of extraction. Processing of PvChi4 was not detected in incompatible interactions with a nonhost strain of F. solani and in symbiotic interactions with Glomus mosseae, and thus may be important only in compatible interactions with F. solani.

Amino Acid Sequence↗
PubMed1995

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.

Amino Acid Sequence↗
PubMed1995

A microsatellite marker for studying the ecology and diversity of fungal endophytes (Epichloë spp.) in grasses.

Randomly amplified polymorphic DNA fingerprinting, which is based on PCR with arbitrary 10-nucleotide primers, were used to analyze genetic diversity among isolates of the endophytic ascomycete Epichloë typhina, which were collected at a single field site from a population of one of its hosts, the grass Bromus erectus. One of the polymorphic randomly amplified polymorphic DNA PCR products occurred in all isolates as single bands with different but closely related sizes. Two of the size variants of this product were cloned and sequenced, and they were found to represent the same DNA sequence, except for a stretch of tandem repeats of the trinucleotide AAG.TTC, which differed in size, consisting of 8 and 18 repeats, respectively. Tandem repeats of this type are called microsatellites. Oligonucleotides were synthesized corresponding to portions of the sequence flanking the microsatellite and were used for PCR amplification of the loci from the genomic DNAs of different Epichloë isolates. A single PCR product was found for most isolates, indicating that the sequence represented a single genetic locus. Five alleles that could clearly be distinguished in size were found in a population of 91 field isolates. PCR with (AAC)8 and (AAG)8 as primers yielded a number of amplified bands from genomic DNA of Epichloë isolates, indicating that these types of microsatellites occur frequently in the genome of this fungus. A survey of all fungal DNA sequences currently deposited in the DNA sequence databases of EMBL and GenBank revealed that microsatellites of different repeating units are widespread in fungi.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1995

Colonization of Transgenic Tobacco Constitutively Expressing Pathogenesis-Related Proteins by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae.

We studied the effect of constitutive expression of pathogenesis-related proteins (PRs) in tobacco plants on vesicular-arbuscular mycorrhiza. Tobacco lines genetically transformed to express various PRs constitutively under the control of the cauliflower mosaic virus 35S promoter of tobacco were examined. Immunoblot analysis and activity measurements demonstrated high levels of expression of the PRs in the root systems of the plants. Constitutive expression of the following acidic isoforms of tobacco PRs did not affect the time course or the final level of colonization by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae: PR-1a, PR-3 (=PR-Q), PR-Q(prm1), PR-4, and PR-5. Similarly, constitutive expression of an acidic cucumber chitinase, of a basic tobacco chitinase with and without its vacuolar targeting peptide, of a basic (beta)-1,3-glucanase, and of combinations of PR-Q and PR-Q(prm1) or basic chitinase and basic (beta)-1,3-glucanase did not affect colonization by the mycorrhizal fungus. A delay of colonization by G. mosseae was observed in tobacco plants constitutively expressing the acidic isoform of tobacco PR-2, a protein with (beta)-1,3-glucanase activity.

Journal Article↗
PubMed1994

Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

Bacterial Proteins↗
PubMed1994

The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant.

In the yeast Saccharomyces cerevisiae, accumulation of the non-reducing disaccharide trehalose is triggered by various stimuli that activate the heat-schock response. Several studies have shown a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. In this study, we have examined mutants defective in genes coding for key enzymes involved in trehalose metabolism with respect to the heat-induced and stationary-phase-induced accumulation of trehalose and the acquisition of thermotolerance. Inactivation of either TPS1 or TPS2, encoding subunits of the trehalose-6-phosphate synthase/phosphatase complex, caused an inability to accumulate trehalose upon a mild heat-shock or upon initiation of the stationary phase and significantly reduced the levels of heat-induced and stationary-phase-induced thermotolerance. Deletion of NTH1, the gene coding for the neutral trehalase, resulted in a defect in trehalose mobilization during recovery from a heat shock which was paralleled by an abnormally slow decrease of thermotolerance. Our results provide strong genetic evidence that heat-induced synthesis of trehalose is an important factor for thermotolerance induction. In an accompanying study [Hottiger, T., De Virgilio, C., Hall, M. N., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 187-193], we present evidence that the function of heat-induced trehalose accumulation may be to increase the thermal stability of proteins.

Acclimatization↗
PubMed1994

The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro.

In baker's yeast (Saccharomyces cerevisiae), accumulation of the non-reducing disaccharide, trehalose, is triggered by stimuli that activate the heat-shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 179-186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose-6P-dehydrogenase, phosphoglucose-isomerase) as well as enzymes of non-yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat-induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose-6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.

Acclimatization↗
PubMed1993

Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity.

Preparations of the trehalose-6-phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively. Recently, we have cloned the gene for the 56-kDa subunit of this complex (TPS1) and found it to be identical with CIF1, a gene essential for growth on glucose and for the activity of trehalose-6-phosphate synthase. Peptide sequencing of the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV. This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa. The N-terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100-kDa subunit, were identical with specific regions of the deduced amino acid sequence. Thus, the sequence cloned represents TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex. Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence. Disruption of TPS2 had no effect on trehalose-6-phosphate synthase activity but caused complete loss of trehalose-6-phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose-6-phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo. These results suggest that TPS2 codes for the structural gene of the trehalose-6-phosphate phosphatase. Heat shock induced an increase in trehalose-6-phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose-6-phosphate phosphatase is subjected to transcriptional control under heat-shock conditions.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.

Amino Acid Sequence↗
PubMed1992

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.

Glucose↗
PubMed1992

Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation.

Trehalose-6-phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose-6-phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose-6-phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose-6-phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

Amino Acid Sequence↗