The plant hormone ethylene is perceived by a five-member family of receptors related to the bacterial histidine kinases. The Raf-like kinase CTR1 functions downstream of the ethylene receptors as a negative regulator of ethylene signal transduction. CTR1 is shown here to be associated with membranes of the endoplasmic reticulum in Arabidopsis as a result of its interactions with ethylene receptors. Membrane association of CTR1 is reduced by mutations that eliminate ethylene receptors and by a mutation in CTR1 that reduces its ability to bind to the ethylene receptor ETR1. Direct evidence that CTR1 is part of an ethylene receptor signaling complex was obtained by co-purification of the ethylene receptor ETR1 with a tagged version of CTR1 from an Arabidopsis membrane extract. The histidine kinase activity of ETR1 is not required for its association with CTR1, based on co-purification of tagged ETR1 mutants and CTR1 after expression in a transgenic yeast system. These data demonstrate that CTR1 is part of an ethylene receptor signaling complex in Arabidopsis and support a model in which localization of CTR1 to the endoplasmic reticulum is necessary for its function. Additional data that demonstrate a post-transcriptional effect of ethylene upon the expression of CTR1 suggest that production of ethylene receptor signaling complexes may be coordinately regulated.

The ethylene receptor ETR1 of Arabidopsis contains transmembrane domains responsible for ethylene binding and membrane localization. Sequence analysis does not provide information as to which membrane system of the plant cell ETR1 is localized. Examination by aqueous two-phase partitioning, sucrose density-gradient centrifugation, and immunoelectron microscopy indicates that ETR1 is predominantly localized to the endoplasmic reticulum. Localization of ETR1 showed no change following a cycloheximide chase. Ethylene binding by ETR1 did not affect localization to the endoplasmic reticulum, based upon analysis of plants treated with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and by examination of a mutant receptor that does not bind ethylene. Determinants within the amino-terminal half of ETR1 are sufficient for targeting to and retention at the endoplasmic reticulum. These data support a central role of the plant endoplasmic reticulum in hormone perception and signal transduction.Hormone perception is necessary for the coordinated growth and development of multicellular eukaryotes. Subcellular localization of hormone receptors is strongly dependent upon the biochemical nature of the hormone (1, 2). For example, polypeptide hormones do not diffuse across membranes and thus utilize receptors localized to the plasma membrane (PM).1 In contrast, steroids can diffuse across membranes and utilize soluble nuclear receptors. In plants, the gaseous hormone ethylene regulates growth, ripening, senescence, abscission, and wound responses (3). Ethylene is diffusible in both aqueous and lipid environments, and a receptor for ethylene theoretically could be localized anywhere within the cell (3).Some constraints upon localization are dictated by the nature of the hormone-binding site. In the plant Arabidopsis, the ethylene receptor family is composed of five members: ETR1, ERS1, ETR2, ERS2, and EIN4 (4). Of these receptors, the ethylene receptor ETR1 has been characterized in most detail because it was the first member of the receptor family identified (5, 6). Analysis of the primary amino acid sequence of ETR1 indicates that there are three predicted transmembrane domains located near the amino terminus: a GAF domain, a histidine kinase domain, and a receiver domain. GAF domains are involved in cGMP binding and light regulation in other proteins, but the function of the GAF domain in ETR1 is unknown (7). Histidine kinase and receiver domains are signaling elements originally identified as components in bacterial phosphorelays and are now also known to be present in plants, fungi, and protists (8). Histidine kinase activity has been demonstrated for ETR1 (9), but the function of this activity in ethylene signal transduction has not been resolved.The predicted transmembrane domains of ETR1 apparently serve two functions. First, they result in membrane localization of the receptor (10). Second, genetic and biochemical evidence indicates that the ethylene-binding site is located within the transmembrane domains of...

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.

Genetic studies in Arabidopsis have provided evidence that ethylene perception in plants is mediated by a family of receptors, including the ETR1 gene product. The ETR1 gene encodes a protein with homology to the twocomponent His kinase regulators that control a variety of signaling cascades in prokaryotic systems and some eukaryotic systems (Chang et al., 1993). While ETR1 was the first ethylene receptor to be identified in plants (Bleecker et al., 1988), additional screens for ethylene-insensitive seedlings and cloning by sequence similarity indicate that additional genes mediate ethylene sensitivity in Arabidopsis (Hua et al., 1995(Hua et al., , 1998 Sakai et al., 1998). The ERS1, ETR2, EIN4, and ERS2 genes all show a high degree of sequence similarity to ETR1 and appear to comprise a small family of ethylene receptors. Dominant point mutations that confer ethylene insensitivity in planta have been isolated in the ETR1, ETR2, and EIN4 genes, and all of these mutations are located within the putative transmembrane domains in the N-termini of these genes. Similar mutations introduced into ERS1 and ERS2 also confer dominant insensitivity when transformed into Arabidopsis plants (Hua et al., 1995(Hua et al., , 1998. These studies indicate that a single mutation in any one of these five genes is sufficient to render plants insensitive to ethylene throughout the plant.Subsequent biochemical experiments have confirmed that the ETR1 gene encodes an ethylene receptor. The N-terminal hydrophobic domain of the ETR1 protein binds ethylene with high affinity when expressed in yeast (Schaller and Bleecker, 1995). The ethylene-binding (sensor) domain of ETR1 consists of three putative membranespanning subdomains that are modeled as alpha helices (Rodriguez et al., 1999). Notably, the etr1-1 mutation in subdomain 2 abolishes ethylene binding by the yeastexpressed protein (Schaller and Bleecker, 1995). Biochemical studies demonstrated that a copper ion in the N-terminal hydrophobic domain of ETR1 is required for ethylene binding, and that the etr1-1 mutation abolishes the capacity of the receptor to coordinate this ion (Rodriguez et al., 1999).Genetic evidence indicates that the ETR1 receptor family signals through the Raf-like kinase CTR1. Loss-of-function mutations in CTR1 show a constitutive triple-response phenotype, indicating that CTR1 acts as a negative regulator of ethylene-response pathways (Kieber et al., 1993). Recently, Hua and Meyerowitz (1998) demonstrated that combining loss-of-function mutants in three or more members of the ETR1 family also results in plants with a constitutive ethylene-response phenotype. These results favor a model for receptor signaling in which the ETR1 receptor family acts in conjunction with CTR1 to suppress response pathways in the absence of ethylene. Ethylene binding would convert receptors to a non-signaling state, resulting in derepression of the response pathway.Based on the concept of ethylene as a negative regulator of the ETR1 receptor family, we hypothesized that domin...

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