Ethylene Controls Autophosphorylation of the Histidine Kinase Domain in Ethylene Receptor ETR1

Molecular Plant

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Volume 1

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Number 2

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Pages 380–387

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March 2008

RESEARCH ARTICLE

Ethylene Controls Autophosphorylation of the Histidine Kinase Domain in Ethylene Receptor ETR1 Jan Voet-van-Vormizeele and Georg Groth1 Heinrich-Heine-Universita¨t, Biochemie der Pflanzen, Universita¨tsstr. 1, 40225 Du¨sseldorf, Germany

ABSTRACT Perception of the phytohormone ethylene is accomplished by a small family of integral membrane receptors. In Arabidopsis, five ethylene receptor proteins are known, including ethylene resistant 1 (ETR1). The hydrophobic aminoterminal domain of these receptors contains the ethylene-binding site while the carboxyl-terminal part consists of a histidine kinase domain and a response regulator domain, which are well known elements found in bacterial two-component signaling. The soluble membrane-extrinsic carboxyl-terminal part of the receptor, which is likely to play an important role in signal transduction, showed intrinsic kinase activity when expressed and purified on its own. However, a correlation between signal input and autokinase activity was not established in these studies, as receptors were missing the transmembrane amino-terminal sensor domain. Thus, it is still unclear whether autophosphorylation occurs in response to perception of the ethylene signal. Here, we report on autophosphorylation studies of purified full-length ETR1. Autokinase activity of the purified receptor is controlled by ethylene or by ethylene agonists like the p-acceptor compound cyanide. In fact, both signal molecules were able to completely turn off the intrinsic kinase activity. Furthermore, the observed inhibition of autophosphorylation in ETR1 by both molecules could be prevented when the ethylene antagonist 1-methyl-cyclopropene (MCP) was applied. Key words: ethylene pathway; phytohormone receptor; membrane protein; two-component system; signal transduction; Arabidopsis thaliana.

INTRODUCTION The gaseous hormone ethylene regulates many aspects of growth and development in plants such as seed germination, seedling growth, leaf and petal abscission, organ senescence, ripening and responses to stress and pathogens. Isolation of mutants in the plant Arabidopsis affecting ethylene responses has led to the identification of ethylene receptors and several downstream components in the ethylene signal transduction pathway (Bleecker and Kende, 2000; Stepanova and Ecker, 2000). According to these studies, ethylene signaling is initiated by a family of integral membrane receptors named ethylene resistant1 (ETR1), ethylene response sensor1 (ERS1), ethylene resistant2 (ETR2), ethylene response sensor2 (ERS2), and ethylene insensitive4 (EIN4), which share evident sequence homology with bacterial and eukaryotic two-component signaling kinases. Sequence analysis suggests that the receptor family classifies further into two subfamilies. ETR1 and ERS1, featuring a well conserved bacterial histidine kinase domain, form subfamily I, whereas ETR2, ERS2, and EIN4, containing an additional transmembrane segment and a degenerate kinase domain, are part of subfamily II. Of the five receptor isoforms

found in Arabidopsis, ETR1 has been characterized in most detail in the past. Sequence analysis suggests that ETR1, like many bacterial histidine kinases, is composed of an aminoterminal sensor domain and a catalytic transmitter domain, but also contains a carboxyl-terminal response regulator domain, which forms an individual element in bacterial twocomponent signaling (Chang, 1996; Urao et al., 2000; Hwang et al., 2002; O’Malley and Bleecker, 2003), making the ethylene receptor a hybrid-type histidine kinase. According to signaling in typical two-component systems, signal perception by the amino-terminal transmembrane domain of ETR1 is supposed to control autophosphorylation of a conserved histidine residue in the catalytic transmitter domain. Then, the phosphoryl group is transferred from histidine to a conserved aspartate in

1 To whom correspondence should be addressed. E-mail: georg.groth@ uni-duesseldorf.de, fax (49)-211-8113569.

ª The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssn004, Advance Access publication 11 February 2008

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the carboxyl-terminal receiver domain of the receptor. From here, the phosphate is transferred to a histidine-containing phosphotransfer protein and is finally located to a response regulator protein, which may activate genes or other transcription factors in the nucleus (Grefen and Harter, 2004). Similar signaling mechanisms are assumed for other plant hybrid-type histidine kinases like cytokinin receptors CKI1 (Kakimoto, 1996) and CRE1 (Inoue et al., 2001) or the putative osmosensor AtHK1 (Urao et al., 1999). In contrast to these receptors, the ethylene receptor proteins are thought to be negatively regulated, namely active in the absence of the plant hormone and inactive in the presence of ethylene (Hua and Meyerowitz, 1998). However, the generality of the phosphorelay mechanism in plants for all hybrid-type histidine kinases was questioned, as characteristic histidine kinase motifs are missing in some of the receptors (Hua et al., 1998; Sakai et al., 1998). Furthermore, experiments on recombinant, truncated forms of the ethylene receptor proteins from Arabidopsis and tobacco (Nicotiana tobacum) lacking the amino-terminal sensor domain have shown phosphorylation on the conserved histidine in case of ETR1, while other ethylene receptors showed predominant phosphorylation on serine or threonine residues (Gamble et al., 1998; Xie et al., 2003; Zhang et al., 2004; Moussatche and Klee, 2004). However, none of the previous studies has addressed the basic question of whether autophosphorylation in the kinase domain of the receptors is related to signal perception in the transmembrane senor domain, which is a fundamental functional characteristic of two-component signaling, as it connects signal perception to signal transduction. The reason why a comprehensive demonstration of an ethylene-triggered kinase activity of ETR1 or any of its isoforms is still missing is owed to the fact that expression and purification of a full-length ethylene receptor containing the transmembrane sensor domain have failed in the past. However, now we have been able to purify the full-length ETR1 receptor protein and to study its intrinsic kinase activity under various conditions. Here, we show that autophosphorylation of recombinant ETR1 is inhibited in response to ethylene or the ethylene agonist cyanide. Further studies showed that the inhibition of autophosphorylation induced by ethylene could be repressed by 1-methyl-cyclopropene (MCP)—an ethylene antagonist broadly used as a ripening inhibitor in agriculture. These results demonstrate a strict interconnection of signal input and signal transduction in the ethylene receptor proteins and emphasize the relevance of the autophosphorylation activity of the receptor in ethylene signal transduction.

RESULTS AND DISCUSSION To study the interdependence of signal input and signal transduction in the ethylene receptor proteins, we have expressed and purified the full-length ETR1 receptor protein and the ETR1165–738 receptor lacking the hydrophobic transmembrane

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sensor domain. Both proteins were solubilized from E. coli cells using 8-M urea and purified by Ni-NTA affinity chromatography followed by a renaturation step to convey the unfolded recombinant proteins in their native structure. Similar refolding protocols have been successfully applied in the past to purify membrane proteins from inclusion bodies, such as the porin OmpC (Kumar and Krishnaswamy, 2005) or the human voltage-dependent anion channel (HVDAC1 and HVDACII) (Engelhardt et al., 2007). A single band on SDS-PAGE confirmed purity and homogeneity of the recombinant ETR1 and ETR1165–738 (Figure 1A). Identity of the proteins was confirmed using antibodies directed against the histidine-tags in both proteins (Figure 1B), as well as an ETR1-specific antibody (Schaller et al., 1995) (Figure 1C). Folding of the recombinant ETR1 proteins was verified by CD spectroscopy. The CD spectra of ETR1 and ETR1165–738 (Figure 2) both show the characteristics of typical a-helical proteins. ETR1 displays minima at 209 and 219 nm, while ETR1165–738 has two minima at 210 and 219 nm, respectively, and a maximum at 195 nm. Both minima are indicative of a predominately a-helical structure of the recombinant proteins (Johnson, 1990; Woody, 1995). Secondary structure calculations by k2d (Andrade et al., 1993; Greenfield, 1996)—a neuronal network algorithm for the estimation of secondary structure composition of an unknown CD spectrum—result in an a-helical content of 40% for ETR1 and of 29% for ETR1165–738 (Table 1). Both figures agree with the numbers predicted from the primary structure of the transmembrane domain of ETR1 (65% a-helices), the known structure of the ETR1 receiver domain (amino acid 604–738) solved by x-ray crystallography (45% a-helices) (Muller-Dieckmann

Figure 1. SDS PAGE and Immunoblot Analysis of Highly Purified, Recombinant ETR1 and ETR1165–738. Purity of ETR1 (lane 1) and ETR1165–738 (lane 2) was confirmed by SDS–PAGE (A). Identity of both recombinant proteins was verified in immunoblot analysis by antibodies directed against the aminoterminal deca-histidine tag (B) and with HRR antibodies directed against the carboxyl-terminal domain (residues 400–738) of ETR1, kindly provided by A. Bleecker, University of Wisconsin, USA) (C).

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Figure 2. CD Spectra of Purified, Recombinant ETR1 and ETR1165–738. CD spectra of ETR1 (red line) and ETR1165–738 (blue line) were obtained by averaging 15 spectra using 0.5-nm intervals at 20 nm min 1. Proteins were applied at a concentration of 0.1–0.2 mg ml 1 and CD data were adjusted to molar ellipticity (H) considering concentration and molar mass of the protein samples.

Table 1. Secondary Structure Elements Calculated from CD Spectra of Recombinant ETR1 and ETR1165–738. a-helix (%)

b-sheet (%)

Random (%)

ETR1

40

20

40

ETR1165–738

29

23

52

et al., 1999) and known structures of soluble bacterial histidine kinases (35–40%) (Tanaka et al., 1998; Tomomori et al., 1999; Ho et al., 2001; Bilwes et al., 1999, 2001), which predict a ahelical content of 42% for ETR1 and 35% for ETR1165–738 in total. The negative regulation of ethylene responses observed in planta suggests that ETR1 exists in its active state in absence of the plant hormone. In order to test the level of purified recombinant ETR1 in its active state, we have analyzed the intrinsic kinase activity of the purified full-length receptor protein by studying autophosphorylation in the absence of ethylene. Phosphorylation was monitored by an in vitro assay using radioactive labelled [c-32P]ATP. As shown in Figure 3A, marked autophosphorylation of ETR1 was observed only in the presence of manganese ions, while, in the presence of other divalent cations like magnesium and calcium ions, no phosphorylation of the receptor was evident. We found the same pattern for autophosphorylation of recombinant ETR1165–738 lacking the transmembrane ethylene sensor domain (Figure 3B). These results agree well with previous studies on truncated forms of the Arabidopsis ETR1 receptor demonstrating that the intrinsic kinase activity of the receptor critically depends on certain divalent cations (Gamble et al., 1998). Maximum phosphorylation was observed in the presence of manganese ions, no or weak autophosphorylation was detected with magnesium or calcium ions. Previous studies

on recombinant, truncated forms of ETR1 have identified a conserved histidine as phosphorylation site (Gamble et al., 1998; Moussatche and Klee, 2004). On the other hand, requirement of histidine kinase activity for ethylene signaling was questioned by mutagenesis studies in planta (Wang et al., 2003; Gamble et al., 2002). However, analysis of mutants containing an inactivated or eliminated histidine kinase domain in a etr1;etr2;ein4 triple mutant background suggest that the histidine kinase domain might play a role in the repression of ethylene response (Qu and Schaller, 2004). None of the mutagenesis studies has addressed the phosphorylation status of the mutant receptors. Thus, it might well be that alternative phosphorylation sites in the protein are addressed when phosphoryl transfer to the canonical histidine is hampered. The principle possibility of serine phosphorylation in a canonical or a degenerated histidine kinase was demonstrated by invitro studies on GST-fusion proteins of ERS1 (subfamily I) and ETR2, ERS2, and EIN4 (subfamily II) (Moussatche and Klee, 2004) and is further supported by the fact that phytochrome photoreceptors have a histidine kinase domain related to bacterial two component systems but show serine/threonine kinase activity (Fankhauser et al., 1999). Together with the analysis of the recombinant proteins by circular dichroism, the results obtained in our in vitro phosphorylation studies strongly support the successful refolding of both proteins during purification. The fact that marked phosphorylation is only observed in the presence of manganese ions for both the truncated and the full-length form of the receptor reveals that not only the isolated soluble kinase domain of the receptor, but also the full-length protein, is capable of autophosphorylation in absence of the signaling molecule ethylene. Previous studies on ETR1 revealed high-affinity binding of ethylene to the amino-terminal sensor domain of ETR1 (Schaller and Bleecker, 1995; Rodriguez et al., 1999). However, binding of the plant hormone was not correlated with the autophosphorylation activity of the receptor in those studies. To address this problem and to confirm functional homology of signal perception and signal transduction in the plant hybridtype histidine kinases, we have determined autophosphorylation of the purified full-length ETR1 in the presence of ethylene or the ethylene agonist cyanide. Like other p-acceptor compounds, cyanide, which was used because of its higher solubility compared to the gaseous compound ethylene, mimics ethylene action and response in planta (Sisler, 1977) and was shown to efficiently compete with ethylene binding in living cyanide-resistant tissue (Solomos and Laties, 1974). Our experiments indicate that presence of ethylene (2 mM) or cyanide (2 mM) completely abolished autophosphorylation of ETR1 (Figure 4A), providing the essential cofactor copper is added to the recombinant receptor. If ethylene, cyanide or copper were applied on their own, no inhibition of ETR1 autophosphorylation was detected. We performed the same experiments with the truncated ETR1165–738 receptor protein, lacking the membrane domain with the ethylene-binding site. As shown in Figure 4B, neither

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Figure 3. Autokinase Activity of ETR1 and ETR1165–738. Effect of divalent cations on ETR1 (A) or ETR1165–738 (B) autophosphorylation (upper panel). Autophosphorylation of purified proteins (0.5 mg ml 1) was studied in 50 mM Tris-HCl, pH 7.5, 100 mM potassium chloride, containing 0.2 mM [c-32P]ATP (1.5 Ci mmol 1 ATP). As cofactors, 10 mM magnesium chloride (lane 2), 10 mM manganese chloride (lane 3) or 10 mM calcium chloride (lane 4) were applied. A control without metal ions but with 1% SDS is shown in lane 1. Reactions were incubated at 20
C for 30 min. and stopped by adding fourfold SDS loading buffer. Reactions were run on 12% SDS-PAGE and blotted to Nitrocellulose. Loaded protein amounts were confirmed by reversible Ponceau S staining of the membrane (lower panel). [c-32P]-labelled ETR1 was visualized and quantified by Fuji FLA 3000 reader (Tokyo, Japan) using Image Gauge software (Fuji, Tokyo, Japan).

Figure 4. Autokinase Activity of ETR1 and ETR1165–738 Associated with Ethylene and the Ethylene Agonist Cyanide. In vitro phosphorylation of purified ETR1 (A) and ETR1165–738 (B) monitored in the presence of ethylene, cyanide and the co-factor copper. Additionally to 10 mM manganese chloride and 0.2 mM [c-32P]ATP (1.5 Ci mmol 1 ATP), protein samples were incubated either with 50 lM copper chloride (lane 1), 2 mM potassium cyanide (lane 2), 2 mM ethylene (lane 3), 50 lM copper chloride, and 2 mM potassium cyanide (lane 4) or 50 lM copper chloride and 2 mM ethylene (lane 5). Equal protein load was verified by Ponceau S staining of the membrane (lower panel).

ethylene, cyanide nor a combination of both molecules with the cofactor copper induced a reduced autophosphorylation of the soluble ETR1 kinase domain. These results strongly suggest functional interconnection of the sensor domain and the transmitter kinase domain in the full-length ethylene receptor, as already demonstrated for typical bacterial two-component systems like NarX (Lee et al., 1999) or PhoQ (Montagne et al., 2001). To further address the functional interconnection of sensor and kinase domain in the ETR1 receptor, we have examined the effect of the ethylene antagonist 1-methyl-cyclopropene (MCP), which inhibits ethylene-induced ripening and senescence in planta (Hall et al., 2000), on ETR1 autophosphorylation. However, even at high concentrations, the antagonist showed no reduction of the intrinsic autokinase activity of ETR1. To clarify whether the intrinsic autokinase activity of ETR1 is really not affected by the binding of MCP or whether

autophosphorylation retained in the presence of MCP is merely related to an inadequate binding of the antagonist to the receptor, we have tested whether MCP is able to revert cyanide-induced inhibition and restore ETR1 autophosphorylation. Indeed, reduced autophosphorylation activity of ETR1 induced by a treatment of the receptor protein with cyanide was restored to basal level after MCP incubation (Figure 5B), suggesting that both compounds compete for the same highaffinity ethylene-binding site in the sensor domain of the receptor. To further confirm that binding of MCP has no effect on the intrinsic kinase activity of the receptor, we have also analyzed quenching of MCP-related autophosphorylation of ETR1 by the ethylene agonist cyanide. Consistent with the results obtained after pre-treatment of ETR1 with cyanide, we observed constantly lower level of cyanide reduced autophosphorylation in the presence of MCP than in the absence of the antagonist (Figure 5C).

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Figure 5. Autokinase Activity of ETR1 Associated with the Ethylene Antagonist 1-MCP. (A) In vitro phosphorylation of recombinant ETR1 at 1 mM 1-MCP (lane 2), at 200 lM cyanide (lane 3), and in untreated control (lane 1, upper panel). (B) Recovery of cyanide-induced inhibition of ETR1 autophosphorylation by 1-MCP. Pre-treatment of recombinant ETR1 with 100 lM cyanide (lane 1) for 10 min followed by the addition of 1 mM 1-MCP (lane 2). (C) Cyanide-induced inhibition of ETR1 autophosphorylation in the absence (lane 1) and presence (lane 2) of 500 lM 1-MCP. Pre-treatment with 1-MCP for 10 min, then 100 lM cyanide was added. All samples also contained 10 mM manganese chloride, 100 lM copper chloride, and 0.2 mM [c-32P]ATP (1.5 Ci mmol–1 ATP). Blot analysis using anti-ETR1 polyclonal antibody HRR and confirming equal protein amount are shown in the lower panels.

In contrast to most animal hormone and growth factor receptors, where binding of the hormone or growth factor results in the activation of the receptor, the ethylene receptors are negatively regulated and exist in an active signaling state in the absence of ethylene, but are converted to an inactive state in the presence of the gaseous phytohormone (Hua and Meyerowitz, 1998). The molecular process controlling the activation state of the receptor might be phosphorylation in the histidine kinase domain—but whether the phosphorylated form is related to the active or inactive state of the receptor has remained unclear. The inhibition of ETR1 autophosphorylation by ethylene and the ethylene agonist cyanide observed in our in vitro assay now suggests that the active state of ETR1 is represented by the phosphorylated form of the receptor and autokinase activity is actually what represses the ethylene response, while inactivation is mediated by a repression of ETR1 autophosphorylation. Antagonists like MCP seem to arrest the receptor in the active phosphorylated form and thereby delay typical ethylene responses like abscission of plant organs, senescence or ripening. Taken together, our results demonstrate strict interdependence of signal perception and autophosphorylation in the ethylene receptor protein and confirm a functional interconnection of sensor and transmitting kinase domain in plant hybrid-type histidine kinases. In this respect, plant ethylene receptors behave like typical bacterial two-component systems. Hitherto, homology with these systems was mainly deduced based on sequence analysis. Now, functional homology in signal-dependent phosphorylation of both systems was verified as well. This result suggests that downstream signaling is accomplished by a phosphorelay mechanism, as suggested by yeast two-hybrid studies (Urao et al., 2000) and recent fluorescence polarization studies in our lab. On the other hand, the ethylene-controlled phosphorylation of ETR1 demonstrated in this study might also control the inter-

action of ETR1 with the soluble kinase CTR1 (Clark et al., 1998), which, in turn, might control downstream signaling of ethylene (Kieber et al., 1993). Further biochemical studies on protein level will be necessary to discriminate these alternative pathways. However, it might well be that both signaling modes are employed by ETR1, depending on internal or external conditions of the plant. To date, analysis of signal transduction by the ethylene receptor family was mainly concentrated on a molecular genetic approach. Now, an in vitro phosphorylation assay on purified recombinant full-length ETR1 established in our lab will allow further detailed screening for new agonists and antagonists of the plant hormone, which are expected to be suitable for many commercial applications in agriculture and floriculture, like extending the vase life of cut flowers or regulating the ripening of fruits and vegetables. In addition to phenotype analysis, the in vitro approach might provide a valuable tool to recognize mutants deficient in signal-dependent phosphorylation and thereby offer great promise for further detailed molecular understanding of (plant) hybrid-type histidine kinases.

METHODS Strains and Growth Conditions Strain BL21 (DE3) (Stratagene, Amsterdam, The Netherlands) and derivate strains C41 (DE3), C43 (DE3) (Miroux and Walker, 1996) of Escherichia coli were used as hosts for expression experiments and strain XL1-Blue of Escherichia coli was used as host for cloning experiments. Cells were grown at 30 or 37
C in 2YT (Maniatis et al., 1989). Where appropriate, ampicillin was added to 150 mg ml 1. Cell density was monitored by measuring absorbance at 600 nm using a Beckmann DU640 spectrophotometer. Solid media were supplemented with 1% (w/v) agar agar.

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Vectors, Plasmids and Nucleic Acid Manipulations Standard recombinant DNA techniques were performed, as described in Maniatis et al. (1989). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Ipswich, UK) and used with their own buffers. PCR reactions were carried out with the PWO-Polymerase kit from PeqLab (Erlangen, Germany). Preparation of plasmid DNA and gel extraction of DNA fragments were performed using QIAprep spin miniprep kit and QIAquick gel extraction kit from Qiagen (Hilden, Germany). For expression, vector pET16b (Novagen, Madison, USA) containing an amino-terminal dekahistidine sequence and a Factor Xa cleavage site was used.

Cloning, Expression and Purification of ETR1 in Escherichia coli To express ETR1 with an amino-terminal dekahistidine-tag, pET16b-ETR1 (construction described in Voet-van-Vormizeele and Groth, 2003) was transformed into strain C43 (DE3). Cultures were grown at 30
C to an optical density of 1 and then induced with 0.5 mM isopropyl b-D-thiogalactopyranosid (IPTG). After 7 h of induction, ETR1 cells were harvested and the cell pellet was re-suspended in buffer B (50 mM Tris-HCl, pH 8.0, 15% (w/v) glycerol, 200 mM NaCl, 0,002% (w/v) phenylmethylsulfonyl fluoride (PMSF)). The solution was twice passed through a French pressure cell at 12 000 psi. The lysate was centrifugated at 30 000 g for 30 min and the pellet was resuspended in buffer C (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 8 M urea). Membrane proteins were extracted by stirring at 37
C for 1 h. Afterwards, the extract was cleared by centrifugation at 30 000 g. After adding imidazole to a final concentration of 20 mM, the ETR1 containing supernatant obtained after solubilization was applied to a Ni-NTA affinity column (0.5 cm diameter 3 7 cm length) previously equilibrated with buffer C. Bound ETR1 was washed with 30 column volumes of buffer C containing 50 mM imidazole and eluated with six column volumes of buffer C containing 250 mM imidazole. The obtained ETR1 receptor was concentrated by Amicon Ultra15 filtrators to a protein concentration of 0.8 mg ml 1. Purified ETR1 was refolded by diluting 1:20 in refolding buffer R1 (50 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.1 mM EDTA, 0.055% PEG 3350, 10 mM DTT, 0.1% (w/v) b-dodecylmaltoside) and ultrafiltrated again. To remove any remaining urea, imidazole and reagents required for refolding, ETR1 was loaded onto a G25 gel filtration column (1 cm diameter 3 4 cm length) and eluated with buffer D containing 50 mM TrisHCl, pH 7.5, 100 mM KCl, 0.1% (w/v)b-dodecylmaltoside and 0.002% PMSF.

Cloning, Expression and Purification of ETR1165–738 in Escherichia coli pET16b-ETR1165–738 was constructed using pET16b-ETR1 as a template for PCR reaction. The restriction sites NdeI and BamHI necessary for cloning the gene into pET16b were added by using primer 1 5#-CCCGGATCCTTACATGCCC-3’ and primer 2

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5#AGCCATATGACTACACTTGTT-3’ in PCR reaction. The correct gene sequence was confirmed by Seq Lab (Go¨ttingen, Germany). ETR1165–738 was expressed in strain BL21 (DE3) containing the plasmid pRARE that is encoding for rare tRNAs. Cells were grown at 37
C and induction was started by adding 0.5 mM IPTG when cells had reached an optical density of 0.7. Cells were harvested 4 h after induction. After re-suspending the cell pellet containing expressed ETR1165–738 in buffer L6 (100 mM BisTris-HCl, pH 6.0, 150 mM NaCl, 8 M urea, 20 mM b-mercaptoethanol, 0.002% (w/v) PMSF), cells were broken by passing twice through a French pressure cell at 12 000 psi. The lysate was centrifugated at 40 000 g for 20 min and the supernatant was diluted with the same volume of buffer L9 (100 mM BisTris-HCl, pH 9.0, 150 mM NaCl, 8 M urea). The resulting protein solution was applied to a NiNTA affinity column (0.5 cm diameter 3 7 cm length) that was equilibrated with buffer L75 (100 mM BisTris-HCl, pH 9.0, 150 mM NaCl, 8 M urea, 0.002% (w/v) PMSF). After stirring the protein solution with Ni-NTA for 60 min, the slurry was washed with 10 column volumes of buffer L75 containing 30 mM imidazole. ETR1165–738 was then eluated with three column volumes of buffer L75 containing 200 mM imidazole. ETR1165–738 elution fractions were concentrated by Amicon Ultra-15 filtrators to a volume of 2.5 ml and then incubated with 10 mM N-ethylmaleimide (NEM) for 10 min in order to alkylate reduced cysteine residues that could disturb the refolding process by forming non-specific disulfide bonds. To remove excess NEM, the protein solution was applied on a G25 gel filtration column (1 cm diameter 3 4 cm length) and eluted with L75 buffer. Purified ETR1165–738 protein was refolded by diluting 1:20 in refolding buffer R2 (50 mM Tris-HCl, pH 8.2, 2 mM CaCl2, 2 mM MgCl2, 10 mM NaCl, 1 mM KCl, 0.002% (w/v) PMSF, 0.03% (w/v) b-dodecylmaltoside (w/v)) and ultrafiltrated again. After diluting twice and concentrating again, the ETR1165–738 was adjusted to a concentration of 2 mg ml 1.

CD Spectroscopy Measurements were performed at room temperature with a Jasco model 720 spectropolarimeter (Jasco Corporation, Tokyo, Japan), using 1-mm-path-length cylindrical cells. A wavelength scan was obtained by averaging 15 spectra using 0.5-nm intervals at 20 nm min 1. All measurements were carried out in 10 mM potassium phosphate, pH 7.4 and 0.03% (w/v) b-dodecylmaltoside at a protein concentration of 0.1–0.2 mg ml 1. Quantitative secondary structure assignments were carried out using k2d (Andrade et al., 1993).

Immunodetection of ETR1 Equal amounts of purified ETR1 and ETR1165–738 were loaded onto an SDS gel containing 12% polyacrylamide. The separated proteins were transferred onto Nitrocellulose (Protran BA83 0.2 mm, Schleicher and Schuell) using a Pharmacia LKB Multiphor II semidry blotting system. Protein transfer was confirmed by reversible Ponceau S staining. The unspecific binding capacity of the membrane was blocked with 1% (w/v) casein in

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TBS buffer containing 50 mM Tris, pH 7.4 and 150 mM NaCl. All further washing steps were performed in TBS-T buffer (50 mM Tris, pH 7.4, 150 mM NaCl and 0.05% (v/v) Tween 20). Commercially available poly-histidine antibodies or the specific anti-ETR1 polyclonal antibody HRR (kindly provided by A. Bleeker, University of Wisconsin, USA) and a secondary antibody coupled to horseradish peroxidase were diluted 1:1000, 1:5000 and 1:10 000, respectively, in TBS buffer with 1% (w/v) casein. Detection of ETR1 was carried out using BM Chemiluminescence Western blotting reagents (Roche, Grenzach-Wyhlen, Germany). Luminescence reaction was detected by Luminescent image analyzer LAS 1000 (Fuji Film, Tokyo, Japan).

Autophosphorylation Assay Autophosphorylation of the recombinant proteins was analyzed in 50 ll reaction buffer (50 mM Tris, pH 7.5, 100 mM KCl, 10 mM MnCl, 0.1% (w/v) b-dodecylmaltoside) containing 0.5 mg ml 1 ETR1 or ETR1165–738, respectively. In experiments addressing the metal ion specificity, manganese chloride was substituted by magnesium chloride or calcium chloride. In experiments studying the effect of ethylene or cyanide on autophosphorylation, 50 lM copper chloride was added to the reaction mixture. Ethylene was supplied by saturating reaction buffer with gaseous ethylene to a concentration of 4.6 mM (OECD Screening Information DataSet, International Programme on Chemistry Safety—INCHEM, Canada). Final concentrations of 2 mM were applied for both effector molecules, ethylene and cyanide. Studies dealing with the ethylene antagonist 1-methylcyclopropen (MCP) were performed with 1 mM MCP and 0.2 mM cyanide to assure excess of MCP towards cyanide. A dextran-linked MCP reagent releasing the gaseous MCP (maximum solubility 2 mM) when dissolved in water was used in these experiments (Rohm and Haas Company, Frankfurt, Germany). Phosphorylation reactions were started by adding 0.2 mM ATP (1.5 Ci mmol 1 ATP). Reactions were incubated for 30 min at 20
C and stopped by adding four-fold SDS loading buffer (100 mM Tris-Borate, pH 8, 17% (w/v) sucrose, 6.5% (w/v) SDS, 0.16% (w/v) bromophenol blue, 50 mM dithiothreitol (DTT)). Reactions were analyzed on 12% SDS-PAGE and transferred to Nitrocellulose (Protran BA83 0.2 mm, Schleicher and Schuell). Protein transfer was confirmed by reversible Ponceau S staining or immunodetection. Phosphate incorporation was visualized by autoradiography.

Analytical Methods Protein concentrations were determined using the bicinchoninic acid method (Pierce, Rockford, USA) using BSA as a standard. Proteins were analyzed by SDS-PAGE on 12% polyacrylamide gels by the Laemmli system (Laemmli, 1970). After electrophoresis, proteins were detected by silver staining according to Heukeshoven and Dernick (Heukeshoven and Dernick, 1988).

ACKNOWLEDGMENTS This work was supported by the Deutche Forschungsgemeinschaft (DFG GR1616/7). No conflict of interest declared.

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