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M A Venis

Publications and source records attributed to M A Venis.

At least 19 recordsLinked to original sources
PubMed2000

Overexpression of auxin-binding protein enhances the sensitivity of guard cells to auxin.

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.

Blotting, Northern↗
PubMed2000

Binding studies.

Explore the source record for details and available documents.

Centrifugation↗
PubMed1998

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH.

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pHi) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pHi, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152-163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1.

Animals↗
PubMed1997

Retention of maize auxin-binding protein in the endoplasmic reticulum: quantifying escape and the role of auxin.

The localisation of maize (Zea mays L.) auxin-binding protein (ABP1) has been studied using a variety of techniques. At the whole-tissue level, tissue printing indicated that ABP1 is expressed to similar levels in all cells of the maize coleoptile and in the enclosed leaf roll. Within cells, the signals from immunofluorescence and immunogold labelling of ultrathin sections both indicated that ABP1 is confined to the endoplasmic reticulum (ER), none being detected in either Golgi apparatus or cell wall. This distribution is consistent with targeting motifs in its sequence. These observations are discussed with reference to the various reports which place a population of ABP1 on the outer face of the plasma membrane, including those suggesting that it is necessary on the cell surface for rapid, auxin-mediated protoplast hyperpolarisation. We have tested the ER, namely that auxin binding induces a conformational change in ABP1 leading to concealment of the KDEL retention motif. Using double-label immunofluorescence the characteristic auxin-induced rise in Golgi-apparatus signal was found, yet no change in the distribution of the ABP1 signal was detected. Maize suspension cultures were used to assay for auxin-promoted secretions of ABP1 into the medium, but secretion was below the limit of detection. This can be ascribed at least partly to the very active acidification of the medium by these cells and the instability of ABP 1 in solution below pH 5.0. In the insect-baculovirus expression system, in which cell cultures maintain pH 6.2, a small amount of ABP1 secretion, less than 1% of the total, was detected under all conditions, Insect cells were shown to take up auxin and no inactivation of added auxin was detected, but auxin did not affect the level of ABP1 in the medium. Consequently, no evidence was found to support the model for auxin promotion of ABP1 secretion. Finally, quantitative glycan analysis was used to determine what proportion of ABP1 might reach the plasma membrane in maize coleoptile tissue. The results suggest that less than 15% of ABP1 ever escapes from the ER as far as the cis-Golgi and less than 2% passes further through the secretory pathway. Such leakage rates probably do not require a specialised mechanism allowing ABP1 past the KDEL retrieval pathway, but we are not able to rule out the possibility that some ABP1 is carried through associated with other proteins. The data are consistent with the presence of ABP1 both on the plasma membrane and in the ER. The relative sizes of the two pools explain the results obtained with immunofluorescence and immunogold labelling and illustrate the high efficiency of ER retention in plants.

Animals↗
PubMed1996

Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays).

Methods for the purification and separation of peptidyl prolyl cis-trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appears as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).

Amino Acid Isomerases↗
PubMed1995

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.

Journal Article↗
PubMed1995

Regulation of synthesis and turnover of maize auxin-binding protein and observations on its passage to the plasma membrane: comparisons to maize immunoglobulin-binding protein cognate.

Electrophysiological experiments have indicated that a fraction of the major auxin-binding protein (ABP1) of maize (Zea mays L.) might be a receptor on the outer surface of the plasma membrane. The predominant location of ABP1 is in the lumen of the endoplasmic reticulum (ER), in accord with its C-terminal KDEL retention signal. Little is known about the biology of the protein in vivo or the rate at which it might pass to the cell surface. We have examined the turnover of ABP1 by in vivo labelling of maize coleoptile sections. After different chase times, ABP1 was immunoprecipitated from detergent-solubilised membrane preparations. Two polypeptides coprecipitated with ABP1. Neither was recognised by any ABP1 antibodies nor by monoclonals to ER retention sequences. The possible significance of these coprecipitating polypeptides is discussed. In addition, we have used a monoclonal antibody to precipitate HDEL proteins from the same membrane preparations. Two dimensional electrophoresis and N-terminal sequencing showed that the major HDEL protein precipitated was a member of the heat-shock-protein 70 family, a homologue of BiP (immunoglobulin-binding protein). We have investigated the turnover of this BiP homologue for comparison with ABP1 and found that both had extended lifetimes, with half-lives greater than 24 h. Use of cordycepin to inhibit transcription indicated that ABP1 mRNA was also long-lived. Synthesis of ABP1 was strongly reduced by heat stress, was reduced a little in response to dithiothreitol and was not markedly changed by tunicamycin. In contrast, BiP synthesis increased markedly in response to tunicamycin and dithiothreitol and increased a little after heat stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Authentic processing and targeting of active maize auxin-binding protein in the baculovirus expression system.

The major auxin-binding protein (ABP1) from maize (Zea mays L.) has been expressed in insect cells using the baculovirus expression system. The recombinant protein can be readily detected in total insect cell lysates by Coomassie blue staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our data suggest that ABP1 is processed similarly in both insect cells and maize. The signal peptide is cleaved at the same position as in maize and the mature protein undergoes tunicamycin-sensitive glycosylation, yielding a product with the same mobility on SDS-PAGE as authentic maize ABP1. On immunoblots the expressed protein is recognized by anti-KDEL monoclonal antibodies. Immunofluorescence localization demonstrates that it is targeted to and retained in the endoplasmic reticulum of insect cells in accordance with its signal peptide and KDEL retention sequence. The expressed ABP1 also appears to be active, since extracts of insect cells expressing ABP1 contain a saturable high-affinity 1-naphthylacetic acid-binding site, whereas no saturable auxin-binding activity is detected in extracts from control cells.

Amino Acid Sequence↗
PubMed1992

Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity.

The major auxin-binding protein in maize membranes is thought to function as a physiological receptor. From earlier information, including the use of site-directed irreversible inhibitors, several of the amino acids likely to form part of the active auxin-binding site were provisionally assigned. Inspection of the amino acid sequence of the auxin-binding protein showed a short region containing all but one of these amino acids. We find that antisera raised against a synthetic peptide encompassing this region recognize all isoforms of the maize auxin-binding protein together with homologous polypeptides in other species. We further find that the antibodies hyperpolarize protoplast transmembrane potential in an auxin-like manner. We conclude that these antibodies display auxin agonist activity and that we have identified an essential portion of the auxin-binding site.

Amino Acid Sequence↗
PubMed1992

Epitope mapping reveals conserved regions of an auxin-binding protein.

There is now good evidence that maize (Zea mays) auxin-binding protein (ABP) functions as a receptor. We have synthesized sequential overlapping hexapeptides to map the epitopes recognized by a number of antisera to ABP. Only a few regions of the protein are recognized, and these are shown to be exposed on the surface. Three epitopes predominate, and these are clustered around, but do not include, the glycosylation site. A comparison is made between these maps of sera against purified ABP, maps of sera raised against recombinant maize ABP expressed in Escherichia coli and computer antigenicity predictions. Our anti-(maize ABP) serum recognizes ABP counterparts in other plant species. We have used immunoblotting to affinity-purify the immunoglobulins which cross-react from the antiserum. Epitope mapping of these immunoglobulins suggests that two of the three predominant epitopes may be conserved in both monocotyledonous and dicotyledonous plants. The possible functional significance of these conserved epitopes is discussed.

Biological Evolution↗
PubMed1991

From auxin-binding protein to plant hormone receptor?

Plant scientists have long been expecting the description of hormone receptor proteins from plants. A putative auxin receptor has now been purified and sequenced and we are beginning to discover how the protein functions.

Indoleacetic Acids↗
PubMed1991

Auxin-binding protein--antibodies and genes.

Of several auxin-binding systems that have been characterised the auxin-binding protein (ABP) of maize coleoptile membranes is the best candidate for a true auxin receptor. ABP, which exists as a homodimer of 22 x 10(3) M(r) glycosylated subunits, has been purified, and monoclonal and polyclonal antibodies raised against it. Electrophysiological studies with antibodies indicated the presence of a functional population of auxin receptors on the exterior face of the plasmalemma; electrophysiological experiments with impermeant auxin analogues now reinforce this conclusion. An epitope mapping kit has been used to identify the major epitopes recognised by antibody preparations. Three major epitopes, bracketing the glycosylation site, have been identified in the polyclonal serum. They are also represented in antisera produced in other laboratories and are conserved in ABP prepared from other plants. One monoclonal antibody recognises an epitope close to the amino terminus of ABP and two others recognise the carboxy terminus. The latter antibodies have been used in a sandwich ELISA to demonstrate that auxin binding induces a conformational change in ABP. Maize ABP is encoded by a small gene family and cDNA and genomic clones have been isolated. With a single exception, predicted amino acid sequences indicate remarkably little heterogeneity. The exceptional cDNA sequence predicts 87% amino acid homology with the major class of proteins. Four introns are apparent in the sequence of a complete ABP gene; their sequences are very highly conserved in an incompletely-cloned second gene lacking the first exon. The major difference between the two genes lies in the length of the first intron, which has been estimated to exceed 5.2 kb in the incomplete gene. The site of initiation of transcription has not been unambiguously identified in the complete gene, and some evidence suggests that there may be an additional intron. Homology to maize ABP cDNA has been detected in the genomes of Arabidopsis, spinach and strawberry but not in that of tobacco. A sequence located within the 3'-half of the maize cDNA is highly repeated in the strawberry genome, from which clones with homology to both halves of the maize cDNA (i.e. putative ABP genes) have been isolated.

Amino Acid Sequence↗
PubMed1990

Characterisation of auxin receptors.

Many auxin-binding systems, both membrane-bound and soluble, have been studied, but most lack credibility as receptors. The exception and best candidate as a valid auxin receptor is the auxin-binding protein of maize coleoptile membranes. This protein can be readily solubilised from the membranes and has been purified to homogeneity. It is a glycosylated homodimer of 22 kDa subunits. Preparations of minimum 50% purity were used to immunise rats and five monoclonal antibodies have been derived. Two of these can be mapped to epitopes within a C-terminal 1 kDa region, while the epitope of a third is within 7 kDa of the N-terminus. Immunotitration of receptor abundance in different tissues of maize seedlings shows that roots contain 40-fold less receptor protein per gram of fresh weight than coleoptiles. Pure receptor was produced by native PAGE and used to generate a high titer polyclonal antiserum in rabbits, capable of detecting receptor protein from as little as 1 mg of coleoptile tissue. The polyclonal specifically precipitates the 22 kDa polypeptide from maize membrane extracts, concomitant with removal of auxinbinding activity. The antiserum also detects homologous polypeptides in maize supernatant and in several other species, both monocots and dicots. In some cases, differences in chromatographic behaviour or size have been found. An auxin-induced conformational change in the receptor has been detected with a sandwich ELISA. The receptor gene has been cloned in four laboratories and the sequence data with knowledge of antibody epitopes can be used to identify parts of the protein involved in conformational changes (and perhaps auxin action). We are currently raising antibodies to a polypeptide thought to be part of the auxin binding site. Most of the auxin-binding protein is found in the endoplasmic reticulum but electrophysiological evidence, using polyclonal antiserum and impermeant auxin conjugates on protoplasts, suggests that a small population of functional receptor is accessible at the exterior face of the plasma membrane. Evidence bearing on the localisation and structure of the receptors is discussed.

Antibodies↗
PubMed1989

Partial purification of a protein from maize (Zea mays) coleoptile membranes binding the Ca2+-channel antagonist verapamil.

A protein that binds the calcium-channel antagonist verapamil has been partially purified from maize (Zea mays) coleoptile membranes. The protein was solubilized with the detergent CHAPS ([ 3-(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate) and purified by a combination of ion-exchange, gel-filtration and hydrophobic-interaction chromatography. This resulted in a 120-fold purification. SDS/polyacrylamide-gel-electrophoretic analysis of the polypeptides from the final purification step indicated that the verapamil-binding protein may have a major component of Mr 169,000. The dissociation constants for specific binding of [3H]verapamil to crude and CHAPS-solubilized maize coleoptile membrane fractions are 72 nM and 158 nM respectively, with respective binding-site concentrations of 135 pmol/mg of protein and 78 pmol/mg of protein. In both cases the Scatchard plots are linear, indicating a single class of binding sites. [3H]Verapamil binding to crude maize coleoptile membrane fractions could not be displaced by unlabelled desmethoxyverapamil or by nifedipine, but could be displaced by unlabelled methoxyverapamil.

Kinetics↗
PubMed1988

Separation of calmodulin from calcium-activated protein kinase using calcium-dependent hydrophobic interaction chromatography.

To determine whether a Ca2+-activated protein kinase is regulated by calmodulin, it is necessary to separate it from endogenous calmodulin and from protein kinase activity that is not calcium dependent. We describe here a procedure for achieving these goals using Ca2+-dependent hydrophobic interaction chromatography on phenyl Sepharose in combination with a pH change. The procedure is based on the observation that while calmodulin solubilized from apple fruit membranes binds to phenyl Sepharose in a Ca2+-dependent fashion at both pH 7.0 and 8.5, Ca2+-activated protein kinase from the same source only shows a Ca2+-dependent interaction above pH 7.5. The implications of this finding for the regulation of this Ca2+-activated protein kinase are briefly discussed.

Calcium↗
PubMed1983

A chemoreceptive bilayer lipid membrane based on an auxin-receptor ATPase electrogenic pump.

Auxin-binding proteins have been extracted from coleoptiles and primary leaves of maize and diffusion--reconstituted in phosphatidyl choline/partially-oxidized cholesterol membranes. Measurement of membrane ion flux at 25 mV external potential with buffered KCl electrolyte was performed for the receptor support matrix and various combinations of ATP, receptor and naphthalene-1-acetic acid. Addition of the three components in any order results in a substantial increase in current with a limit-of-detection for auxin of about 10(-7)M. The pH-dependence of the response is consistent with previous suggestions that an ATPase pump acts to translocate protons in the presence of K+ and Mg2+ and that the pump can be activated by auxin. This work provides the first direct link between the binding of a plant hormone to a putative receptor and the evocation of a biochemical response.

Adenosine Triphosphatases↗