This chapter discusses the photoaffinity labeling and strategies for plasma-membrane-protein purification. For the purification of membrane proteins solubilized with detergents, the same methods can be applied as those used to purify soluble proteins; these include gel filtration, ion-exchange, reversed-phase, and affinity chromatography. Auxins play a major role in the regulation of plant cell elongation, cell division, morphogenesis, and differentiation. The molecular sequence of events responsible for the mode of action of auxins is not well understood. Receptor-like proteins that bind auxin and transmit the auxin signal have been postulated. Because of the low concentration of most of these proteins, conventional approaches to identify and characterize them for molecular studies have been fraught with technical difficulties. To overcome problems associated with traditional auxin-binding studies, photolabile synthetic auxins may provide a valuable tool with which to tag auxin-binding proteins. Photoaffinity probes have contributed greatly to the identification of receptor proteins and have aided in structural studies.

A 23 kDa protein (p23) was identified in microsomal extracts from maize coleoptiles by photoaffinity labelling with 5-azido-[7-3H]indol-3-ylacetic acid ([3H]N3IAA). Labelling of p23 was blocked by unlabelled IAA, N3IAA, indol-3-ylbutyric acid and indol-3-yl-lactate. In addition, labelling was efficiently decreased by tryptophan, as well as by the scavenger p-aminobenzoic acid. Labelling was, however, not affected by synthetic auxins such as 1-naphthylacetic acid or 2,4-dichlorophenoxyacetic acid. Competition data suggest that the label was probably bound via the indole ring, and hence labelling was not specific for auxins. The 23 kDa protein was solubilized from crude microsomes by extraction with Triton X-100 and purified to homogeneity by ion-exchange, size-exclusion and reversed-phase chromatography. After electroblotting, the amino acid sequences of the p23 N-terminus as well as the several tryptic peptides were obtained. Database comparisons revealed sequence identity with a maize manganese superoxide dismutase. We conclude that photoaffinity labelling of p23 was pseudo-affinity, and therefore the binding site for IAA is not specific.

Two cAMP-binding proteins, cbp1 and cbp2, were purified from the cytoplasm of the green alga Volvox carteri. Both proteins have a native molecular mass of 90 kDa as determined by gel filtration. cbp2 was purified to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDS/PAGE. The cbp1 preparation contains a 42-kDa and a 44-kDa band. The cAMP-binding activity is not associated with protein kinase activity. Tryptic peptides of cbp2 were sequenced by automated Edman degradation. Two pairs of peptides differ in one amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the cAMP-specific phosphodiesterases of V. carteri with respect to charge, molecular mass and binding affinity to N6-cAMP-agarose. Reverse-phase chromatography of the bound ligand revealed that the two binding proteins hydrolyse cAMP to 5' AMP. The binding specificity of purified cbp1 and cbp2 was probed by a set of modified cAMP derivatives. Both proteins bind cAMP strictly specifically in the anti conformation; position 1 and 6 of the adenine moiety and at least one of the exocyclic O atoms of the ribose cyclic phosphate moiety are essential. 3-Isobutyl-1-methylxanthine is an effective inhibitor of binding but the natural methylxyanthines are not. At present it is not clear whether cbp1 and cbp2 are individual proteins or isoforms of one another.

We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, suggesting that both proteins are encoded by different members of a gene family.

Virtually all aspects of plant growth and development are influenced by structurally relatively simple substances termed phytohormones. It has been argued that the wide range of responses elicited by these substances requires a mode of action that is radically different from those of animal hormones. Current evidence indicates that enzymes that can synthesize and modify phytohormones and their antagonists, or hydrolyze phytohormone conjugates to release active hormones, play a role in initiating important regulatory pathways. They are also likely to provide invaluable tools with which to study the mechanisms underlying growth and differentiation in plants. Here we describe recent biochemical progress in the characterization of the molecular targets of phytohormones.

Plants modify gene expression and metabolism in response to a large variety of exogenous and endogenous signals. Among the various signals sensed by plants the phytohormones e.g. auxins, cytokinins, ethylene, abscisic acid and gibberellins, have received particular attention. It has been argued that, similar to hormone action in `vertebrates’ the first step in phytohormone action is the interaction of a ligand with binding sites, most likely proteins, located either at the plasma membrane or at various other intracellular locations. It is thought that binding of phytohormones to such proteins should be specific, reversible, of high affinity and saturable and result in a defined biological response.

A biologically active and photolabile auxin analog, 5‐azido‐[7‐3H]indole‐3‐acetic acid ([3H]N3IAA), was used to search for auxin‐binding proteins in cytosolic extracts from maize coleoptiles (Zea mays L.) and identified a protein with a molecular mass of 60 kDa (p60). Binding of [3H]N3IAA is highly specific as demonstrated by competition analysis with functionally relevant auxin analogs. p60 is found in coleoptiles and roots of etiolated maize seedlings and was detected in cytosolic as well as in microsomal fractions. The protein binds to 1‐naphthylacetic acid (1‐NAA) sepharose and is eluted with auxins. A purification scheme resulting in homogenous p60 protein was devised and it was shown that p60 has β‐d‐glucoside glucohydrolase activity (E.C.3.2.1.21). The hydrolytic activity of p60 for the synthetic substrate p‐nitro‐phenyl‐β‐d‐glucopyranoside is diminished by 1‐NAA. p60 shows high substrate specificity since it hydrolyzes indoxyl‐O‐glucoside, but not β‐(1,4)‐cellobiose, IAA‐inositol or IAA‐amino acid conjugates. The present data suggest that p60 might be involved in the hydrolysis of auxin conjugates.

Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA), we identified several IAA-binding proteins with molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, we were able to selectively extract pm23 from the plasma membrane. We show that auxins and functional analogues compete with [3H]N3IAA for binding to pm23. We found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, we identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. We designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7000-fold purification.

1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier-mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, we have synthesized a tritiated, photolabile NPA analogue, 5'-azido-[3,6-3H2]NPA ([3H2]N3NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of [3H2]N3NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with [3H2]N3NPA and exposure to UV light, we observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-[7-3H]indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.

Conference Article| February 01 1992 Auxin-binding proteins of Zea mays identified by photoaffinity labelling Ian Moore; Ian Moore 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Joachim Feldwisch; Joachim Feldwisch 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Narciso Campos; Narciso Campos 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Rolf Zettl; Rolf Zettl 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Bretislav Brzobohaty; Bretislav Brzobohaty 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Laszlo Bakó; Laszlo Bakó 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Jeff Schell; Jeff Schell 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Klaus Palme Klaus Palme 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linne Weg 10, D-5000 Köln 30, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (1): 70–73. https://doi.org/10.1042/bst0200070 Article history Received: September 19 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Ian Moore, Joachim Feldwisch, Narciso Campos, Rolf Zettl, Bretislav Brzobohaty, Laszlo Bakó, Jeff Schell, Klaus Palme; Auxin-binding proteins of Zea mays identified by photoaffinity labelling. Biochem Soc Trans 1 February 1992; 20 (1): 70–73. doi: https://doi.org/10.1042/bst0200070 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: IAA, indole-3-acetic acid, TIBA, 2,3,5-triiodobenzoic acid, NPA, 1-N-naphthylphthalamic acid, azido-IAA, 5-azido-7-[3H]indole-3-acetic acid, PCR, polymerase chain reaction, NAA, naphthylacetic acid, azido-NPA, 5′-azido-[3,6-3H]NPA This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.

The organisation of growth and development in vascular plants appears to be highly adapted to meet the specific demands of a sessile, autotrophic habit. Many of the characteristic features of plant development are associated with the activities of five groups of phytohormones. Each of the phytohormones has the ability to influence fundamentally a remarkable variety of developmental and physiological processes. This ability has been widely documented but remains to be explained. Here we describe how recent breakthroughs in the analysis and understanding of eucaryotic signal transduction are being applied, in conjunction with technical advances in molecular genetics, to elucidate the molecular basis of the phytohormonal properties of auxin. Both auxin concentration, and the sensitivity of plant cells to this phytohormone have been implicated as important parameters in auxin action. We describe recent molecular biological approaches to assess the contribution made by each of these parameters. Emphasis is given to a description of recent genetic and biochemical progress towards identification of the molecular targets of the auxin signal and the molecular components involved in its subsequent transduction.

Oncogenes carried by the transferred DNA (T‐DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T‐DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T‐DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28‐fold increase in conversion of tryptophan to indole‐3‐lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis‐regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax‐1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole‐3‐acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.

To understand precisely the mechanisms by which hormones like auxins regulate plant differentiation and development, it is essential to isolate putative hormone receptors. We have purified the major auxin binding protein from maize coleoptiles to homogeneity. The protein has an apparent molecular weight of 22,000 Da and binds 1-naphthylacetic acid with a KD of 2.4 x 10(-7) M. Protein sequence analysis allowed the construction of oligonucleotide probes to isolate a corresponding cDNA coding for this protein. The open reading frame of this cDNA predicts a protein of 201 amino acids and 21,990 Da in size. The amino acid sequence includes a cleavable N-terminal signal sequence and a C-terminal signal element consisting of the amino acids Lys Asp Glu Leu known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum.

In eucaryotic cells external signals detected by receptors are translated into a limited repertoire of intracellular second messengers. Occupancy of these receptors initiates the production of active messengers, including the well studied cyclic adenosine monophosphate (cAMP) as well as the recently discovered messenger molecules that are derived from phosphoinositides such as arachidonic acid, inositol-l,4,5-triphosphate, and 1,2-diacylglycerol (for review see: Berridge 1986; Newton and Brown 1986; Boss and Morre 1989). These messengers are capable of regulating a vast array of physiological and biochemical processes either by direct interaction with distinct proteins or indirectly by activating enzymes which trigger conformational changes in the final target proteins. However, the number of second messengers in eucaryotic cells appears to be surprisingly small, indicating that most probably only a limited number of internal signal pathways are needed, albeit remarkably universally in all eucaryotes analyzed up to now, to transduce these signals to their final biological destination.

The major auxin‐binding protein from maize coleoptiles was purified to homogeneity. The protein has an apparent mol. wt of 22 kd and binds 1‐naphthylacetic acid with a KD of 2.40 x 10(‐7) M. Additional antigenically related proteins, present in very low amounts, could be demonstrated in maize coleoptiles using immunodetection. Extensive protein sequence analysis of the major auxin‐binding protein allowed the construction of several synthetic oligonucleotide probes which were used to isolate a cDNA coding for this protein. The cDNA corresponds to a mRNA with a 3′‐poly(A)+ sequence and a single, long open reading frame of 603 bases. The open reading frame, starting 34 residues from the 5′ end of the cDNA, predicts a 21,990 Dalton protein of 201 amino acids. Comparison of this deduced amino acid sequence with the partial amino acid sequences of purified auxin‐binding protein, revealed a perfect match, involving a total of 53 amino acid residues. The primary amino acid sequence includes a 38‐amino‐acid‐long N‐terminal hydrophobic leader sequence which could represent a signal for translocation of this protein to the endoplasmic reticulum. An additional signal is located at the C‐terminal end, consisting of the amino acids KDEL known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum in eucaryotic cells. The primary sequence contains a N‐glycosylation site (‐asp133‐thr‐thr‐). This site was found to be glycosylated by a high‐mannose‐type oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)