©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ethylene Response Mediator ETR1 from Arabidopsis Forms a Disulfide-linked Dimer (*)

G. Eric Schaller (1), Andrea N. Ladd (1)(§), Michael B. Lanahan (2), Jon M. Spanbauer (1), Anthony B. Bleecker (1)(¶)

From the (1) Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 and the (2) Agricultural Group, Monsanto Company, Chesterfield, Missouri 63198

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mutations in the ETR1 gene of the higher plant Arabidopsis confer insensitivity to ethylene, indicating a role for the gene product in ethylene signal perception and transduction. The ETR1 gene product has an amino-terminal hydrophobic domain and a carboxyl-terminal domain showing homology to the two-component signal transduction proteins of bacteria. We report here that in both its native Arabidopsis and when transgenically expressed in yeast, the ETR1 protein is isolated from membranes as a dimer of 147 kDa. Treatment with the reducing agent dithiothreitol converted the dimer to a monomer of 79 kDa, indicative of a disulfide linkage between monomers. Expression of truncated versions of ETR1 in yeast confirmed that the high molecular mass form is a homodimer and demonstrated that the amino-terminal region of ETR1 is necessary and sufficient for this dimerization. Site-directed mutagenesis of two cysteines near the amino terminus of ETR1 prevented formation of the covalently linked dimer in yeast, consistent with a role in disulfide bond formation. These data indicate that ETR1 may use a dimeric mechanism of signal transduction in a manner similar to its bacterial counterparts but with the additional feature of a disulfide bond between monomers.


INTRODUCTION

Ethylene serves as a gaseous hormone in plants. As a plant growth regulator, it is involved in seed germination, seedling growth, leaf abscission, fruit ripening, and organ senescence. In addition, ethylene can mediate the induction of specific defense proteins (reviewed by Abeles et al.(1992)). Although the ethylene biosynthetic pathway is now well characterized (reviewed by Kende(1993)), the means by which cells recognize and transduce the ethylene signal have not been established. An important contribution to our understanding of ethylene signal transduction has come about through the generation of mutants in the plant Arabidopsis thaliana with altered ethylene sensitivity. At least four separate genes in the ethylene signal transduction pathway have been identified using this approach (Bleecker et al., 1988; Guzman and Ecker, 1990; Kieber et al., 1993; Van der Straeten et al., 1993). The cloning of two of these genes, CTR1 (Kieber et al., 1993) and ETR1 (Chang et al., 1993), has recently been reported.

Mutations in ETR1 result in an ethylene-insensitive phenotype in Arabidopsis (Bleecker et al., 1988; Chang et al., 1993). All four of the mutant alleles of ETR1 that have been isolated are dominant to the wild-type gene and affect all ethylene responses examined. Plants containing the mutant etr1-1 allele display only one-fifth of the saturable ethylene binding of that found in wild-type plants, indicating that ETR1 may be directly involved in ethylene perception (Bleecker et al., 1988). Based on this work and the epistatic relationship of ETR1 with other ethylene response mutants (Kieber et al., 1993), mutations of ETR1 affect an early step in the ethylene signal transduction pathway. These findings are consistent with the possibility that ETR1 may be the ethylene receptor or a regulator of the pathway.

The ETR1 gene was cloned and found to encode a polypeptide with homology to the bacterial two-component signal transduction proteins (Chang et al., 1993). In bacteria, these signal transduction systems contain a sensor and a response regulator and mediate responses to a wide variety of environmental stimuli (reviewed in Parkinson(1993)). The sensor protein is typically membrane-localized and contains a histidine kinase domain that autophosphorylates on a conserved histidine residue in response to an environmental stimulus. The phosphate is then transferred to an aspartic acid residue on the response regulator protein, which mediates downstream events. The ETR1 protein of Arabidopsis has both histidine kinase and response regulator domains with the conserved residues required for activity of the bacterial counterparts. It also contains a hydrophobic domain indicative of membrane localization (Chang et al., 1993).

The finding that the ETR1 gene of Arabidopsis (Chang et al., 1993) as well as the SLN1 gene of yeast (Ota and Varshavsky, 1993) encode proteins with homology to the histidine kinases and response regulators of bacteria provides evidence that the two-component signal transduction pathways of bacteria are present in eukaryotes. However, questions remain as to how this system has been adapted for signal transduction in eukaryotes and, more specifically, ethylene signal transduction in plants. In this paper, we report that the product of the ETR1 gene is a dimer in both its native Arabidopsis and when transgenically expressed in yeast. The yeast system has been used to resolve which residues of ETR1 are required for dimerization. The finding that ETR1 forms a dimer has implications as to the manner by which the ethylene signal is transduced in plants.


MATERIALS AND METHODS

Plasmid Constructions

Standard molecular cloning techniques were used (Sambrook et al., 1989; Ausubel et al., 1994). A 4.2-kb()EcoRI fragment of the genomic ETR1 clone (Chang et al., 1993) was used as probe in screening Arabidopsis cDNA libraries. A 2.85-kb cDNA clone (cETR1-9) in pBlueScript containing the complete coding sequence for ETR1 was isolated from an Arabidopsis cDNA library cloned into the EcoRI site of ZAPII (Kieber et al., 1993; library provided by J. Ecker, University of Pennsylvania, Philadelphia). To remove noncoding sequence at the 5`- and 3`-ends of cETR1-9, the clone was used as template DNA for a PCR reaction (Perkin-Elmer Corp. PCR kit) using a 5`-primer (CCCGGATCCATAGTGTAAAAAATTCATAATGG) and a 3`-primer (CCCGGATCCTTACATGCCCTCGTACAGTACCCGG). The PCR product (cETR1-5.2) was digested with BamHI and cloned into the BamHI site of pBlueScript KS-, and the product was confirmed by sequencing (Sequenase kit, U. S. Biochemical Corp.). In addition, a 1.4-kb cDNA clone (cETR1-1) containing partial coding sequence and flanked by EcoRI sites was isolated from an Arabidopsis cDNA library cloned in YES (library provided by R. Davis, Stanford University, Stanford, CA).

For expression of fusion proteins with glutathione S-transferase in Escherichia coli, the vector pGEX-2T (Pharmacia Biotech Inc.) was used. One fusion protein, designated GST-HRR, was designed to express that portion of ETR1 corresponding to amino acids 401-738. For this, the 1.4-kb EcoRI fragment of cETR1-1 was cloned into the EcoRI site of pGEX-2T. A second fusion protein, designated GST-UNK, was designed to express that portion of ETR1 corresponding to amino acids 165-400. For this, a DraI-BglII fragment of cETR1-9 was cloned into the SmaI-EcoRI sites of pGEX-2T, following addition of an 8-mer EcoRI linker to the end-filled BglII site.

For expression in yeast, the vector pYcDE-2 was used (Hadfield et al., 1986). This vector has a constitutive ADC1 promoter, an EcoRI cloning site, and allows for Trp selection. For expression of full-length ETR1, the BamHI fragment of cETR1-5.2 was isolated, end-filled, 10-mer EcoRI linkers were added, and the fragment was cloned into the EcoRI site of pYcDE-2. For expression of the amino-terminal region of ETR1 representing amino acids 1-400, a 1.4-kb EcoRI-BglII fragment of cETR1-9 was cloned into pYcDE-2, following addition of a 10-mer EcoRI linker to the end-filled BglII site. For expression of the region of ETR1 representing amino acids 183-738, a 2.1-kb DraI-EcoRI fragment of cETR1-9 was cloned into pYcDE-2, following addition of an 8-mer EcoRI linker to the DraI site. For this construct, Met-183 was the first ATG codon capable of serving as the initiator for translation.

Site-directed mutants for expression in yeast were constructed as follows. For the etr1-1 mutation (C65Y), a PCR product was first generated from a 4.2-kb EcoRI subclone of the etr1-1 mutant gene (Chang et al., 1993) using the same primers and subcloning procedure as for cETR1-5.2. A BstXI restriction fragment containing the etr1-1 mutation from this subclone was used to replace the corresponding wild-type BstXI fragment in cETR1-5.2. The etr1-1 mutant was then expressed in yeast in the same manner as the full-length wild-type sequence. Site-directed mutants in the cETR1-5.2 clone for C4S and C6S together and for C99S were made using the Altered Sites Mutagenesis System (Promega) according to the manufacturer. The double mutant of C65Y and C99S was made by replacing the 0.9-kb HpaI-BglII wild-type fragment of the etr1-1 (C65Y) mutant with the corresponding fragment of the C99S mutant. All mutations were confirmed by sequencing.

Yeast Transformation, Growth, and Protein Isolation

Constructs were transformed (Schiestl and Gietz, 1989) into yeast strain LRB520 (MAThis3leu2trp1 ura3-52yck2-1::HIS3), and standard media and procedures were used for growth (Ausubel et al., 1994).

For isolation of soluble and membrane fractions, cultures were grown at 30 °C to mid-log phase. All subsequent steps were performed at 4 °C. Cells were harvested by centrifugation at 1,500 g for 5 min, washed in 10 mM Tris (pH 7.5), 0.5 M sucrose, 2.5 mM EDTA, and then resuspended in homogenization buffer (100 mM Tris (pH 7.5), 0.3 M sucrose, 5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin as protease inhibitors at 1 ml of buffer/g of cells. Cells were broken open by vortexing with chilled glass beads (Ausubel et al., 1994), and the homogenate was centrifuged at 10,000 g for 10 min to remove debris and intact organelles. This supernatant was centrifuged at 100,000 g for 30 min to separate the soluble protein fraction from the pelletable membrane fraction. The membrane pellet was resuspended in 10 mM Tris (pH 7.5), 10% (w/w) sucrose, 1 mM EDTA with protease inhibitors.

For analysis of yeast membranes by density gradient centrifugation, total membranes were fractionated on a 20-53% (w/w) sucrose gradient in 10 mM Tris (pH 7.5), 1 mM EDTA. After overnight centrifugation at 30,000 rpm (Beckman rotor SW41Ti), 0.75-ml fractions were collected and stored at -70 °C until use. Activities of mitochondrial cytochrome-c oxidase (Villalba et al., 1992) and endoplasmic reticulum NADH-cytochrome c reductase (Lord, 1987) were measured as described. 1 unit of activity corresponds to the oxidation or reduction of 1 µmol of cytochrome c/min. Presence of the plasma membrane H-ATPase was determined by Western blot analysis, using a polyclonal antibody generated against the carboxyl terminus of the enzyme (Monk et al., 1991; antibody provided by R. Serrano, Universidad Politecnica, Valencia, Spain).

For extraction of total yeast protein into SDS-PAGE loading buffer, 5-ml yeast cultures were grown and isolated in mid-log phase. Cells were pelleted, washed with water, and then resuspended in 75 µl of glucuronidase buffer (50 mM Tris (pH 7.5), 10 mM MgCl, 1 M sorbitol). After the addition of 10 µl of -glucuronidase (Sigma, G7017), cells were incubated 90 min at 30 °C. Cells were washed with glucuronidase buffer and then extracted directly into 75 µl of SDS-PAGE loading buffer.

Isolation of Arabidopsis Membranes

Arabidopsis was grown in liquid culture as described (Chang et al., 1992). Leaves were homogenized at 4 °C in extraction buffer (30 mM Tris (pH 8), 20% glycerol, 5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 10 µg/ml aprotinin as protease inhibitors. The homogenate was strained through Miracloth (Calbiochem) and centrifuged at 8,000 g for 15 min. The supernatant was centrifuged at 100,000 g for 30 min, and the membrane pellet was resuspended in 10 mM Tris (pH 7.5), 10% (w/w) sucrose, 5 mM EDTA with protease inhibitors.

Treatment with Dithiothreitol (DTT)

Protein samples were mixed with SDS-PAGE loading buffer (125 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 0.01% bromphenol blue, 1 mM phenylmethylsulfonyl fluoride) containing 100 mM DTT. Controls contained no DTT. Samples were incubated at 37 °C for 1 h and then subjected to SDS-PAGE (Laemmli, 1970). Boiling samples caused aggregation of the ETR1 protein.

Antibody Production and Western Blotting

Fusion proteins were expressed in E. coli and affinity purified on glutathione-Sepharose (Smith and Johnson, 1988). Antisera were prepared from recombinant proteins by the University of Wisconsin Medical Facility, cleared against E. coli proteins as described (Chang et al., 1992), and used at a 1:1000 dilution.

For Western blotting, protein samples were fractionated on 8% polyacrylamide gels in the presence of SDS and transferred to nitrocellulose. Immunodecorated ETR1 was visualized using a chemoluminescence detection system according to the manufacturer (Kirkegaard and Perry). Densitometric analysis of immunodecorated bands was performed using NIH Image (version 1.6) after first scanning the exposed film using Adobe Photoshop and an Agfa scanner.


RESULTS

Expression of the ETR1 Gene in Yeast

Hydropathy analysis of ETR1 identifies three potential transmembrane segments at the amino terminus (Fig. 1A). Although ETR1 does not contain a canonical signal sequence for membrane insertion, a topological analysis based on the model recognition approach of Jones et al.(1994) predicts that the amino terminus of ETR1 would lie on the extracytoplasmic side of a membrane while the large carboxyl-terminal region containing histidine kinase and response regulator domains would lie on the cytoplasmic side of a membrane (Fig. 1B). The sequence analysis does not provide any information as to which membrane system in the cell the ETR1 protein is localized. Since ethylene is diffusable in both aqueous and lipid environments (Abeles et al., 1992), ETR1 could theoretically be present in any membrane.


Figure 1: ETR1 structure and expressed portions of the sequence. A, hydrophobic, histidine kinase, and response-regulator (RR) domains of ETR1 are indicated. For expression of fusion proteins in E. coli, glutathione S-transferase (GST) is shown as a blackrectangle, with expressed regions of ETR1 (UNK and HRR) indicated by openrectangles. Regions of ETR1 expressed in yeast are indicated by openrectangles. B, predicted transmembrane structure of ETR1 constructs expressed in yeast.



To aid in the analysis of ETR1, two polyclonal antibodies were raised against different regions of the ETR1 protein by expressing those regions as fusion proteins with glutathione S-transferase in E. coli (Fig. 1A). One antibody, designated Ab-UNK, was generated against amino acids 165-400 of ETR1 and covers a region of unknown function. A second antibody, designated Ab-HRR, was generated against amino acids 401-738 of ETR1 and covers most of the putative histidine kinase domain and the entire response regulator domain. In an initial analysis of Arabidopsis extracts, both antibodies were found to recognize several polypeptides in soluble and membrane fractions. An unambiguous assignment of ETR1 to any of these proteins was not possible, prompting us to express the full-length cDNA in yeast. Yeast has proved to be an appropriate system for the functional expression of eukaryotic proteins, in particular membrane proteins that can be difficult to express in E. coli (Villalba et al., 1992). As such, yeast could serve as a model system to address questions concerning the biochemical properties of ETR1.

A cDNA clone containing the entire coding sequence for ETR1 was expressed in yeast using a shuttle vector with constitutive promoter. Soluble and membrane fractions were then isolated from control yeast transformed with just the vector or from yeast transformed with the ETR1 construct. An immunodetectable polypeptide of 79 kDa was identified in yeast expressing ETR1 but not in control yeast transformed with vector alone (Fig. 2). This 79-kDa polypeptide is close to the molecular mass of 82 kDa predicted from amino acid sequence of ETR1. The 79-kDa ETR1 polypeptide was predominantly in the pelletable membrane fraction of yeast, with very little in the soluble fraction (Fig. 2). To determine if ETR1 was present as a membrane protein rather than an insoluble aggregate, the pelletable membrane fraction was further analyzed by sucrose density gradient centrifugation. On the gradient, the ETR1 polypeptide resolved into a discrete vesicle population with a peak at 35% (w/w) sucrose (Fig. 3), confirming that ETR1 is indeed expressed as a membrane protein in yeast, consistent with the hydropathy analysis. The peak of ETR1 did not align precisely with any of the membrane markers examined. In this respect, it should be noted that when the plasma membrane H-ATPase of plants was expressed in yeast, the protein was fully functional, but rather than being exported to the plasma membrane, it was retained in membranes derived from the endoplasmic reticulum (Villalba et al., 1992). This vesicle population peaked at 32% (w/w) sucrose when analyzed by sucrose gradient centrifugation, close to what we observed with the ETR1 protein.


Figure 2: Expression of ETR1 in yeast. Soluble (S) and membrane (M) fractions were isolated from control yeast transformed with vector alone or from yeast expressing ETR1. Protein was reduced in 100 mM DTT, subjected to SDS-PAGE, and ETR1 was visualized by Western blot using two antibodies (UNK or HRR) generated against different domains of ETR1. Migration positions of molecular mass markers are indicated in kilodaltons.




Figure 3: Sucrose density gradient centrifugation of membranes from yeast expressing ETR1. Total membranes were loaded on a linear sucrose gradient, and 0.75-ml fractions were collected after overnight centrifugation to use for marker analysis. A, distribution of plasma membrane H-ATPase, mitochondrial cytochrome c (Cytc) oxidase, and endoplasmic reticulum cytochrome c reductase as yeast membrane markers. B, distribution of wild-type (wt) ETR1 on gradient shown in A and mutant ETR1 (C4S, C6S) from a second gradient run under similar conditions.



Presence of a High Molecular Mass Species of ETR1 in Yeast and Arabidopsis

The ETR1 protein expressed in yeast migrated at one of two different molecular masses on SDS-PAGE, depending upon treatment with reducing agent (Fig. 4). In the presence of the reducing agent DTT, the immunodetectable protein migrated at 79 kDa, while in the absence of DTT, the protein migrated at 147 kDa.


Figure 4: Sensitivity of ETR1 to reductant. Membrane fractions from yeast or Arabidopsis were incubated in the absence (-) or presence (+) of 100 mM DTT for 1 h at 37 °C. Protein was subjected to SDS-PAGE, and ETR1 was visualized by Western blot using antibodies UNK or HRR. Positions of the 147- and 79-kDa species of ETR1 are indicated.



Arabidopsis membranes were examined for ETR1 protein with the same characteristics observed in the heterologous yeast system. In the absence of reducing agent, both domain-specific antibodies immunodecorated a protein of 147 kDa in Arabidopsis membranes (Fig. 4). In the presence of reducing agent, the 147-kDa protein was no longer present and a new protein of 79 kDa appeared. These immunodecorated proteins in Arabidopsis are of identical mass to the high and low molecular mass species of ETR1 expressed in yeast. Although the UNK and HRR antibodies also cross-reacted with additional proteins, none were recognized by both antibodies, and these may represent unrelated proteins or proteolytic fragments of ETR1.

Based upon these results, we conclude that ETR1 is present as a high molecular mass species when isolated from either its native Arabidopsis or the heterologous yeast system and is capable of being converted to a lower molecular mass species by reduction. The requirement of reducing agent for conversion of the high to the lower molecular mass species is indicative of a disulfide linkage. In addition, the oxidized 147-kDa species has approximately twice the molecular mass of the 79-kDa reduced species, consistent with the possibility that it represents a dimer of the reduced species. The primary difference between ETR1 expressed in its native Arabidopsis and the heterologous yeast system is that ETR1 occurred in about 100-fold lower abundance in Arabidopsis membranes than in yeast membranes.

Dimerization of the ETR1 Polypeptide

The heterologous yeast system was used as a model system to further analyze dimerization of ETR1. In addition to the full-length ETR1 protein, two truncated versions of ETR1 were expressed in yeast (Fig. 1). One truncated version, ETR1(1-400), covered the first 400 amino acids of ETR1 and included the amino-terminal hydrophobic domain. The second truncated version, ETR1(183-738), covered amino acids 183 through to the carboxyl terminus and included the entire histidine kinase and response regulator domains. To minimize the possibility that the high molecular mass species of ETR1 was of artifactual origin, a simplified extraction procedure was used to isolate protein from transformed yeast. The cell wall of yeast was digested with -glucuronidase, and then total protein was immediately extracted into SDS-PAGE sample buffer. The apparent molecular mass of ETR1 proteins was determined in the absence and presence of reducing agent (Fig. 5). Both the full-length ETR1(1-738) and the truncated ETR1(1-400) versions of ETR1 exhibited high and low molecular mass species, depending upon the presence of reducing agent. In contrast, the truncated ETR1(183-738) exhibited only one molecular mass whether or not reducing agent was present. These results indicate that the amino-terminal region of ETR1 is necessary and sufficient to form the high molecular mass species. Furthermore, the difference in mass between the high and lower molecular mass species of ETR1(1-738) and ETR1(1-400) was in each case close in value to the mass of the monomer, a finding consistent with homodimer formation and inconsistent with the alternative possibility that ETR1 forms a heterodimer with an unidentified yeast protein.


Figure 5: Sensitivity of full-length and truncated forms of ETR1 to reductant. Total protein was isolated from yeast expressing full-length (amino acids 1-738) and truncated (amino acids 1-400 and 183-738) forms of ETR1. Protein was incubated in the absence (-) or presence (+) of 100 mM DTT for 1 h at 37 °C, subjected to SDS-PAGE, and ETR1 was visualized by Western blot using antibody UNK. Molecular masses of the various ETR1 species are indicated based on standards.



The sensitivity of the ETR1 homodimer to reducing agents is indicative of a disulfide linkage between cysteine residues. In the amino-terminal region of ETR1 there are four candidates for cysteine residues that could be involved in the formation of disulfide bridges. The possible involvement of these cysteine residues for homodimer formation was examined by site-directed mutagenesis. Two cysteine residues (Cys-4 and Cys-6), located in the amino-terminal domain predicted to lie on the extracytoplasmic face of a membrane, were changed together to serine residues. A third cysteine residue (Cys-65) is present in the middle of the second transmembrane segment. The ethylene-insensitive mutant, etr1-1, results from a change of this cysteine to a tyrosine residue and was used for this analysis. A fourth cysteine residue (Cys-99) is present in the middle of the third transmembrane segment and was changed to a serine residue. When expressed in yeast, neither single or double mutants of Cys-65 and Cys-99 abolished the ability of ETR1 to form a covalently linked homodimer (Fig. 6). However, a mutant of ETR1, in which the first two cysteines (Cys-4 and Cys-6) were changed to serine residues, lost its ability to form a covalently linked homodimer (Fig. 6). This mutation does not affect the ability of the ETR1 protein to insert in the membrane because membranes containing the mutant ETR1 still resolved at 35% (w/w) sucrose when analyzed by density gradient centrifugation (Fig. 3), like membranes containing wild-type ETR1. These results indicate that one or both of the initial cysteine residues is necessary for forming the disulfide-linked dimer of ETR1.


Figure 6: Sensitivity of site-directed mutants of ETR1 to reductant. Total yeast protein was isolated, incubated in loading buffer for SDS-PAGE without reducing agent for 1 h at 37 °C, subjected to SDS-PAGE, and ETR1 was visualized by Western blot using antibody UNK. Single letter amino acid codes used are C (Cys), S (Ser), and Y (Tyr).




DISCUSSION

ETR1 from both its native Arabidopsis and a transgenic yeast system has been isolated as a dimer joined by a covalent disulfide linkage. Since ETR1 is capable of forming a dimer in yeast and Arabidopsis, dimerization may be a self-assembly process with a disulfide bond formed following an initial noncovalent association. Otherwise, one must postulate that additional factors needed for association are present in both yeast and Arabidopsis. The results from yeast demonstrate that two ETR1 monomers form a cross-linked homodimer, although this does not preclude the possibility that in Arabidopsis ETR1 could form a heterodimer with an isoform of similar molecular mass (Chang and Meyerowitz, 1995). In any case, the consistency of results observed with ETR1 expressed in Arabidopsis and the heterologous yeast system indicates that yeast may be a suitable system for further analysis of ETR1 structure and function. This finding is of particular importance given that the ETR1 protein appears to be expressed in native plant tissue at very low levels.

Mutagenesis of two cysteines located near the amino terminus of ETR1 prevented formation of the covalent linkage between monomers of ETR1 when expressed in yeast, consistent with a role in disulfide bond formation. These cysteines are located in a portion of ETR1 predicted to be on the extracytoplasmic face of a membrane (Fig. 1). Disulfide bonds have frequently been observed in the extracytoplasmic domains of other proteins, notably dimeric growth factors and the insulin receptor of animals, and a wide variety of proteins involved in the cell wall of both plants and fungi. This is consistent with the localization of protein-disulfide isomerase, an enzyme of the endoplasmic reticulum that catalyzes the formation of disulfide bonds (Noiva, 1994). While the amino-terminal cysteines appear to be both necessary and sufficient for covalent dimerization, we cannot rule out the possibility that formation of disulfide linkages within the membrane may subsequently occur. In addition, we cannot entirely preclude the possibility that a disulfide linkage is formed during protein isolation, although conditions where this is known to occur were avoided (Kumar and Davidson, 1992). Even in such a case, the cysteines would function in cross-linking and still indicate a dimeric structure to ETR1 with the amino termini in close proximity (Pakula and Simon, 1992).

The finding that ETR1 is a dimer indicates that it may function similarly to the bacterial histidine kinases in which the dimer plays a vital role in the signaling process (Parkinson, 1993; Pan et al., 1993). With the bacterial histidine kinases, perception of the environmental signal results in an intermolecular phosphorylation event in which one monomer phosphorylates its neighboring monomer upon a histidine residue. While this dimeric form is apparently in place prior to perception of the signal, in all known cases it is a noncovalent association of monomers (Parkinson, 1993; Pan et al., 1993). The finding that the dimeric form of ETR1 involves a covalent disulfide linkage represents an intriguing variation upon the bacterial paradigm.

The relationship between the biochemical properties of the ETR1 protein and the mechanism of ethylene signal transduction is not yet clear. Evidence presented here that the amino-terminal half of the ETR1 protein is necessary and sufficient for dimerization, in addition to the previous finding that all of the dominant mutations of ETR1 result from single amino acid changes in the hydrophobic domain (Chang et al., 1993), compel us to consider the importance of this dimerization domain in the transduction of the ethylene signal. This region may be particularly important in determining the quaternary structure of the dimer, with ethylene-mediated changes in the structure necessary for propagation of the ethylene signal. Dimerization of ETR1 may help explain the dominant effect of mutations in the gene. If both the mutant homodimer and the mutant/wild-type heterodimer were inactive, then only 25% of the dimers would be expected to be functional in a heterozygous plant. This could be below the limit needed to induce measurable ethylene responses. Alternatively, the dominant ETR1 mutant proteins could be locked in an active state and be unable to respond to changes in the ethylene signal. Both mechanisms for dominance, either inactivation or locking of the dimeric form in one active state, have been previously observed with the bacterial histidine kinases (Pazour et al., 1991; Pan et al., 1993). Interestingly, a mutation that apparently locks the VirA sensor protein into an active conformation also lies within a transmembrane domain of the protein (Pazour et al., 1991). While the bacterial paradigm is useful in developing models for ETR1 function, it should be noted that it has not yet been proven that mutations in ETR1 are of the dominant loss-of-function type as defined by Herskowitz(1987). It is conceivable that these mutations arise due to a gain of function, in which case the true function of ETR1 could be obscured. Further biochemical characterization of ETR1 with regard to kinase activity and direct interactions of the protein with ethylene should help to resolve this issue.


FOOTNOTES

*
This work was supported by Dept. of Energy Grant DE-FG02-91ER20029.000 (to A. B. B.) and by the DOE/NSF/USDA Collaborative Research in Plant Biology Program (Grant BIR92-20331). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Animal Sciences Dept., University of Arizona, Tucson, AZ 85721.

To whom correspondence should be addressed. Tel.: 608-262-4009; Fax: 608-262-7509.

The abbreviations used are: kb, kilobase(s); DTT, dithiothreitol; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Joseph Ecker, Ronald Davis, Ramon Serrano, Lucy Robinson, and Caren Chang for generously providing materials and information, James Siedow for helpful discussions, and Richard Vierstra and Michael Sussman for critical reading of the manuscript.


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