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Two types of association between phytochrome and crude membrane fractions from oat (Avena sativa L.) are distinguished and compared, and that which comprises only a small fraction of the total phytochrome in extracts prepared in the absence of added divalent cations (Watson & Smith, 1982b) has been studied in detail. Extraction in the presence of phenylmethylsulphonly fluoride shows that proteolysis of Pr (the red-light absorbing form) probably does not account for the lower levels of membrane-associated phytochrome measured after far-red light than after red light. Difference spectra of soluble and membrane-associated phytochrome indicate that the latter is much less susceptible to spectral degradation in vitro than is the soluble pool. The stoichiometry of association with the membranes is such that for each phytochrome molecule associated after far-red light there are three associated after red light and it is argued that this stoichiometry is maintained independent of the extraction pH. The characteristics of this photreversible association of phytochrome with membranes are compared to the characteristics of the widely studied light-induced enhancement of phytochrome pelletability that is dependent on electrostatic interaction of phytochrome and membranes. [ABSTRACT FROM AUTHOR]

A series of fluence-response curves for the binding of phytochrome to membranes is the absence of divalent cations, as described by Watson & Smith (1982), were constructed to demonstrate that the response obeys the law of reciprocity. Analysis of the binding of Pfr (the far-red-absorbing form of phytochrome) showed that two Pfr molecules bind to the membrane for each Pr (the form with an absorption maximum in the red) photoconverted to Pfr in the intrinsic membrane-bound phytochrome pool. Using this stoichiometry we have been able to model the binding curve of Pr and match the binding data. Pr binding can be simulated of PR binds only as a consequence of the binding of Pfr, i.e. when Pfr is part of a Pr : Pfr dimmer. The enrichment of the membranes with Pfr as a result of the binding of Pfr was also accurately simulated. There is no binding cooperativity. Phytochrome binding is a low-fluence response and the possibility that it has physiological significance as a mediator of phytochrome action is discussed. [ABSTRACT FROM AUTHOR]

High level expression of the major auxin-binding protein (ABP1) from maize (Zea mays L.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immunolocalization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.

To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and carboxy-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.

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)

The epitopes of two monoclonal antibodies raised to a putative auxin receptor have been mapped. Carboxy-peptidase A digestion of the antigen, auxin-binding protein (ABP) purified from maize, completely abolished binding of antibody MAC 256 and impaired binding of MAC 259, suggesting that they both recognise C-terminal epitopes. Published sequences of ABP showed that the C terminus was KDEL, a tetrapeptide used for targeting proteins to the ER in animal cells. We have used this short homology to confirm that the two monoclonals recognise C-terminal KDEL, showing that animal KDEL proteins and synthetic KDEL peptides are recognised and that animal cell ER is stained strongly and specifically. Sucrose density gradient fractionation of maize microsomal membranes showed that plant KDEL proteins, including ABP, fractionated with markers for the endoplasmic reticulum. However, few proteins are stained by anti-KDEL monoclonals in plants. For comparison, a monoclonal antibody raised to a synthetic HDEL peptide was also used and found to stain a set of proteins in all plant species tested. The anti-HDEL and anti-KDEL monoclonals were sequence specific, staining different proteins. On density gradient fractionation HDEL proteins also banded with ER marker activities. However, the intracellular distribution of HDEL and KDEL proteins determined by immunofluorescence was different. Whereas HDEL proteins showed a distribution characteristic of plant ER, and this localisation was confirmed by immunogold labelling of ultrathin sections and electron microscopy, KDEL proteins showed strong fluorescence in discrete parts of the cell cortex. These observations are discussed in terms of the potential these monoclonal antibodies have as markers for ER and of the role ABP plays in plant cell signalling.

Reacquisition after extinction often appears faster than original acquisition. However, data from conditioned suppression studies indicate that this effect may arise from spontaneous recovery and reinstatement of unextinguished contextual stimuli related to the unconditioned stimulus (US). In the present experiments using the rabbit nictitating membrane preparation, spontaneous recovery was eradicated before reaquisition training. US contextual stimuli were controlled by retaining the US during extinction through explicit unpairings of the conditioned stimulus (CS) and US. Attempts were also made to drive the associative strength of the CS into the inhibitory region by differential conditioning and conditioned inhibition procedures. In all cases, reacquisition was very rapid in comparison with a rest control. The results are discussed with respect to their implications for CS and US processing models of conditioning.

Since Pavlov, theories of conditioning have assumed that CR evocation is governed by a series of internal stimuli generated by the CS. This hypothesis was tested in conditioning of the rabbit's nictitating membrane (NM) response by attempting to manipulate the internal sequence through truncating a delay CS and extending a trace CS on test trials. These perturbations of CS duration produced large deficits in CR likelihood and smaller alterations in the CR's time course. As predicted by many models of conditioning, the onset of the CS appeared to play a large but not exclusive role compared to CS duration and CS offset in both evocation and timing of the CR. The results are discussed with respect to their implications for real-time models of conditioning.

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.

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.

Two experiments examine cross-modal transfer of response features specific to the interstimulus interval (ISI) between a conditioned stimulus (CS) and an unconditioned stimulus. Rabbits were given initial training with a stimulus (CSA) in one modality (e.g., tone) at a designated ISI (e.g., 600 ms). Training was then shifted to a new stimulus (CSB) in another modality (e.g., light) at a new ISI (e.g., 400 ms). The timing of early conditioned responses (CRs) to CSB reflected the ISI of CSA. Ultimately, CRs to CSB shifted to a temporal location conforming to the ISI of CSB. When the ISI of CSB was shorter than that of CSA, CRs to CSA also shifted to a locus conforming to the ISI of CSB. The present results confirmed previous findings that training in one CS modality accelerates CR acquisition to a CS in another modality. The findings are compared with the transfer of response patterns in instrumental learning sets and are discussed regarding their implications for theories of cross-modal transfer.

The monoclonal antibody MAC 256 precipitates specifically the auxin-binding protein (ABP) of maize membranes. Auxin-binding activity was recovered from the immunoprecipitate and MAC 256 can, therefore, bind undenatured, native ABP. A sandwich enzyme-linked immunosorbent assay was used to present native ABP to MAC 256 and under these conditions auxins inhibit antibody binding. Millimolar naphthalene-1-acetic acid completely blocks MAC 256 binding and the characteristics of monoclonal antibody MAC 259 are similar. The ability of a range of auxins and related compounds to displace MAC 256 correlates with the known structure-activity relationships of these compounds in vivo and in binding assays. The results are interpreted in terms of an auxin-induced conformational change in ABP, auxin binding leading to a change in, or concealment of, the epitope of the antibody. The epitope for MAC 256 and 259 lies close to the carboxy terminus of the protein, implying that the part of ABP containing the sequence of amino acids responsible for retention within the endoplasmic reticulum is conformationally active.

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.

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps - anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.

We present a convenient method to calculate the efficiency of fluorescence energy transfer in two-dimensional membrane systems. We apply it to the analysis of energy transfer between phospholipid molecules labelled with fluorescein and rhodamine groups, and of energy transfer in reconstituted membranes containing (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, with the ATPase labelled at the ATP binding site with fluorescein as donor, and rhodamine-labelled lipid as acceptor. The ATP binding site is found to be distant from the plane of the lipid/water interface of the membrane. It is suggested that the ATPase is present in the membrane as a dimer, with the two ATP binding sites in the dimer being close to the protein/protein interface. Addition of vanadate causes no change in quenching, suggesting that the ATP binding site does not move significantly with respect to the lipid/water interface in the E1-E2 conformational transition of the ATPase.

We have studied cross-linking of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum and in reconstituted systems, using glutaraldehyde, cupric-1,10-phenanthroline and 3,3'-dithiobis (sulphosuccinimidylpropionate). All reagents produce extensive cross-linking, forming aggregates too large to enter polyacrylamide gels. Only traces of cross-linked dimeric ATPase species are formed. Saturation transfer electron spin resonance spectra of spin-labelled sarcoplasmic reticulum cross-linked with glutaraldehyde are also consistent with the formation of extensively cross-linked aggregates in the membrane. The results are interpreted in terms of dynamic clusters of ATPase molecules in the membrane, probably in the form of rows of ATPase molecules.