Biomedical subjects

L Sticher

Publications and source records attributed to L Sticher.

14 recordsLinked to original sources

PubMed2001

Protein-binding partners of the tobacco syntaxin NtSyr1.

Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.

Arabidopsis Proteins↗

PubMed2001

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis.

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

Amino Acid Sequence↗

PubMed1997

Systemic acquired resistance.

This paper examines induced resistance (SAR) in plants against various insect and pathogenic invaders. SAR confers quantitative protection against a broad spectrum of microorganisms in a manner comparable to immunization in mammals, although the underlying mechanisms differ. Discussed here are the molecular events underlying SAR: the mechanisms involved in SAR, including lignification and other structural barriers, pathogenesis-related proteins and their expression, and the signals for SAR including salicylic acid. Recent findings on the biological role of systemin, ethylene, jasmonates, and electrical signals are reviewed. Chemical activators of SAR comprise inorganic compounds, natural compounds, and synthetic compounds. Plants known to exhibit SAR and induced systemic resistance are listed.

Journal Article↗

PubMed1993

Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

Amino Acid Sequence↗

PubMed1992

Vacuolar chitinases of tobacco: a new class of hydroxyproline-containing proteins.

The fungicidal type I chitinases contribute to the defense response of plants against pathogens. Two tobacco chitinases represent a different class of hydroxyproline-containing proteins. Hydroxyproline-rich proteins are predominantly extracellular, structural glycoproteins proteins that lack enzymatic activity and contain many hydroxyproline residues. In contrast, type I chitinases are vacuolar enzymes. They are not glycosylated and contain a small number of hydroxyproline residues restricted to a single, short peptide sequence.

Amino Acid Sequence↗

PubMed1992

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins.

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.

Amino Acid Sequence↗

PubMed1992

alpha-Amylase Isoforms are Posttranslationally Modified in the Endomembrane System of the Barley Aleurone Layer.

The subcellular site of the posttranslational modification of alpha-amylase was investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya). Aleurone layers of Himalaya barley synthesize and secrete two groups of alpha-amylase isoforms, referred to as low-isoelectric point (low-pl) or HAMY1 and high-pl or HAMY2, when incubated in gibberellic acid and CaCl(2). Whereas homogenates of aleurone layers contain four isoforms of HAMY1 with pls 4.90, 4.72, 4.64, and 4.56, incubation media contain predominantly isoforms 4.72 and 4.56. Microsomal membranes isolated from aleurone layers contain all four isoforms of HAMY1. Microsomal membranes can be resolved into two peaks by isopycnic density gradient centrifugation: a peak of heavy membranes with endoplasmic reticulum and Golgi apparatus (GApp) marker enzyme activities and a peak of light membranes with characteristics of the GApp. The heavy membranes contain proportionally more HAMY1 pl 4.90 and 4.64 isoforms, whereas light membranes contain a higher proportion of pl 4.72 and 4.56 isoforms. Experiments with the ionophore monensin show that membranes of the GApp as well as the endoplasmic reticulum are involved in the posttranslational modification of HAMY1 isoforms. Monensin inhibits the secretion of alpha-amylase and causes the enzyme to accumulate within the cell. Precursor forms of HAMY1 accumulate in light membranes isolated from monensin-treated aleurone layers indicating that the GApp is involved in the conversion of the precursor to the secreted forms of the enzyme.

Journal Article↗

PubMed1992

In vitro processing of tomato proteinase inhibitor I by barley microsomal membranes: a system for analysis of cotranslational processing of plant endomembrane proteins.

A plant-derived in vitro system for the study of cotranslational processing of plant endomembrane proteins has been developed and used to investigate cotranslational proteolytic processing of tomato proteinase inhibitor I. Translation of the inhibitor I precursor in wheat germ lysate supplemented with barley aleurone microsomal membranes resulted in cotranslational import of the protein into microsomal vesicles and cleavage of the signal sequence. NH(2)-terminal sequence analysis of the translocated inhibitor I processing intermediate showed that the signal sequence was cleaved between Ala(23) and Arg(24) of the precursor protein. Parallel experiments using dog pancreas microsomal membranes indicated an identical site of cleavage, suggesting that the substrate determinants for signal sequence processing are conserved across kingdoms. The plant-derived processing system used for this study may be valuable for analysis of cotranslational processing of other plant preproteins and for characterizing the components of the cotranslational import machinery in plants.

Journal Article↗

PubMed1991

A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.

Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.

Amino Acid Sequence↗

PubMed1991

Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro.

Posttranslational modifications that give rise to multiple forms of alpha-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley alpha-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). alpha-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and alpha-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic alpha-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley alpha-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify alpha-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of alpha-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.

Journal Article↗

PubMed1990

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes.

The effects of heat shock on the synthesis of alpha-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25 degrees C to 40 degrees C for 3 hours, inhibits the accumulation of alpha-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca(2+). When ER is isolated from heat-shocked aleurone layers, less newly synthesized alpha-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca(2+) transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.

Journal Article↗

PubMed1989

The calcium requirement for stability and enzymatic activity of two isoforms of barley aleurone alpha-amylase.

alpha-Amylases (EC 3.2.1.1) secreted by the aleurone layer of barley grains are Ca2+-containing metalloenzymes. We studied the effect of Ca2+ on the activity and structure of the two major groups of aleurone alpha-amylase by incubating affinity purified enzyme in solutions containing Ca2+ from pCa 4 to 7. Both groups of isoforms required one atom of Ca2+/molecule of enzyme as determined by isotope exchange, but the two groups differed by more than 10-fold in their affinity for Ca2+. Both groups of alpha-amylase were irreversibly inactivated by incubation in low Ca2+ (pCa 7). This inactivation was not due to changes in primary structure, as measured by molecular weight, but appeared to be the result of changes in secondary and tertiary structure as indicated by circular dichroism spectra, serology, lability in the presence of protease, and fluorescence spectra. Analysis of the predicted secondary structure of barley aleurone alpha-amylase indicates that the Ca2+-binding region of barley amylases is structurally similar to that of mammalian alpha-amylases. Our data indicate that micromolar levels of Ca2+ are required to stabilize the structure of barley alpha-amylases in the endoplasmic reticulum of the aleurone layer where these enzymes are synthesized.

Calcimycin↗

PubMed1988

Evidence for precursor forms of the low isoelectric point alpha-amylase isozymes secreted by barley aleurone cells.

Gibberellin-treated barley (Hordeum vulgare L.) aleurone cell protoplasts have been shown previously to contain two alpha-amylase isozymes which are not secreted (JV Jacobsen, JA Zwar, PM Chandler 1985 Planta 13: 430-438). This report shows that these intracellular forms are immunochemically related to the low isoelectric point but not the high isoelectric point group of alpha-amylase isozymes and that they arise by new synthesis like the secreted forms. Pulse-chase studies show that the intracellular isozymes are precursors to the secreted isozymes. Conversion of the intra- to the extracellular forms involves decreases in isoelectric points with no change in size detectable by SDS-PAGE. The precursor isozymes were also detected in aleurone layer homogenates but they were unstable. They could be stabilized by various treatments including heating the homogenate to 70 degrees C for 10 minutes indicating that the instability was enzymically mediated. Using purified radioactive precursor isozymes, it was shown that instability did not involve inactivation but the conversion to secreted forms. The nature of the covalent modification associated with conversion was not determined but available data indicate that it does not involve glycosylation.

Journal Article↗

PubMed1981

Calcium requirement for the secretion of peroxidases by plant cell suspensions.

Spinach (Spinacia oleracea, L.) cells in liquid culture release peroxidases. This release is reduced by EGTA and promoted by calcium ions. In a medium deprived of calcium the rate of peroxidase release is low, but immediately after addition of I mM calcium there is a sudden increase of the extracellular peroxidase activity. Extracellular calcium apparently penetrates into the cultured cells rather freely and, as a consequence, the rate of peroxidase secretion by these cells is directly correlated with the concentration of calcium in the medium. Magnesium, at twice the concentration used for calcium, has no effect on the release of peroxidases. Cells treated with Na azide, Na hydrogenarsenate of fluphenazine secrete less peroxidase upon addition of calcium.

Calcium↗