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J. Biol. Chem., Vol. 277, Issue 22, 19861-19866, May 31, 2002

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Localization of the Ethylene Receptor ETR1 to the Endoplasmic Reticulum of Arabidopsis^*

Yi-Feng Chen, Melynda D. Randlett, Jennifer L. Findell, and G. Eric Schaller

From the Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824

Received for publication, February 7, 2002, and in revised form, March 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 ETR1 (6, 11). According to the current model, ETR1 is a disulfide-linked homodimer (10) with each dimer containing a single ethylene-binding site (11). High affinity binding of ethylene is mediated by a copper co-factor that is coordinated by two conserved amino acids (Cys-65 and His-69) (6, 11) present in the second transmembrane domain. The importance of the transmembrane domains is emphasized by the finding that, although the other members of the ethylene receptor family share similar features with ETR1, the highest degree of amino acid conservation is found within this region implicated in ethylene binding (12). The apparent requirement of a hydrophobic environment for optimal ethylene binding may have favored use of a membrane-bound receptor for ethylene signal transduction.

Ethylene perception in plants thus represents an intriguing variation on known ligand-receptor paradigms as it involves a readily diffusible ligand but a membrane-bound receptor. Although some members of the ethylene receptor family have predicted signal sequences, suggesting entry into the secretory system, ETR1 has no signal sequences or obvious information to indicate subcellular localization (10). In this paper, we demonstrate that ETR1 predominantly localizes to the endoplasmic reticulum (ER) of Arabidopsis. Subcellular localization to the ER has implications for ethylene signal transduction and for our understanding of the factors that determine receptor localization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Membrane Fractionation-- Microsomal membranes were isolated from Arabidopsis plants grown in liquid culture as described (10) using homogenization buffer containing 50 mM Tris (pH 8.2), 20% glycerol, 1 mM dithiothreitol, 2 mM EDTA, and protease inhibitors. Aqueous two-phase partitioning was performed using a 6.4% (w/w) Dextran T500/PEG3350 mixture (13). For sucrose density gradient centrifugation, isolated membranes were resuspended in 25 mM Tris (pH 7.5), 10% sucrose, 1 mM dithiothreitol, 2 mM EDTA, and protease inhibitors. The microsomes were then layered onto a 20-50% (w/w) sucrose gradient in 10 mM Tris (pH 7.5), 1 mM dithiothreitol, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. Gradients were centrifuged at 100,000 × g for 16 h, and 1-ml fractions were collected. For analyses performed in the presence of Mg²⁺, 5 mM MgCl₂ was added to homogenization and centrifugation buffers.

Membrane Markers-- Specific membranes were identified by use of antibodies against the ER markers BiP and ACA2 (14-16), the PM marker H⁺-ATPase (17), and the tonoplast marker VM23 (18). Thylakoid membranes were identified by spectrophotometric analysis of chlorophyll levels, and Golgi membranes were identified based on Triton-stimulated UDPase activity (19).

Immunoblot Analysis-- Immunoblot analysis was performed as described (20). Two antibodies were used for detection of ETR1. One antibody termed anti-ETR1-(401-738) was generated against amino acids 401-738 of ETR1 (10) and was used to identify full-length ETR1. A second antibody termed anti-ETR1-(165-400) was generated against amino acids 165-400 of ETR1 (10) and was used to identify the truncated receptor ETR1-(1-349).

Preparation of Monospecific Anti-ETR1 Antibody-- A monospecific anti-ETR1 antibody was prepared from the polyclonal antibody anti-ETR1-(401-738) (10). The serum was depleted of antibodies that cross-react with glutathione S-transferase (GST) by passing through a column of Affi-Gel-10 (Bio-Rad) cross-linked to GST. The antibody was affinity-purified by binding to an Affi-Gel column cross-linked to GST-ETR1-(401-738) (10), then eluting with 0.1 M glycine (pH 2.5). As a final purification step, the antibody was passed through an Affi-Gel column coupled with soluble proteins from Arabidopsis.

Immunogold EM-- Leaf segments of Arabidopsis were fixed with 4% (w/v) paraformaldehyde and 1% (w/v) glutaraldehyde, dehydrated, then embedded in Epon 812 resin, and polymerized for 3 days at 60 °C. Thin sections were cut and collected onto naked nickel grids. Section surfaces were etched with 10% H₂O₂ for 15 min, blocked with 2% (w/v) bovine serum albumin and 0.2 M glycine in PBST (phosphate-buffered saline, 0.02% Tween 20), and then incubated with antibodies in 1% bovine serum albumin/PBST overnight at 4 °C. After washing with PBST, grids were incubated for 1 h with protein A-colloidal gold conjugate (10 nm, Sigma) in 1% bovine serum albumin/PBST, poststained in 5% uranyl acetate for 15 min and lead citrate for 5 min, and then observed with a transmission electron microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biochemical Analysis of ETR1 Localization-- The ethylene receptor ETR1 has a modular structure (Fig. 1A) and contains three predicted transmembrane segments implicated in both membrane localization and ethylene binding (6, 10). We analyzed the localization of ETR1 in Arabidopsis membranes by two independent biochemical strategies: aqueous two-phase partitioning and sucrose density-gradient fractionation. Aqueous two-phase partitioning in polyethylene glycol-Dextran mixtures allows for purification of PM vesicles based on their preferential partitioning into the upper phase (13). Partitioning is dependent upon surface properties of the membranes and under optimal conditions can result in about 90% of the plasma membrane partitioning to the upper phase and 80-90% of the intracellular membranes partitioning to the lower phase (13, 17). Following partitioning of Arabidopsis membranes, the relative distribution between the two phases of ETR1 and various membrane markers was determined by immunoblot analysis (Fig. 1B). The PM marker (H⁺-ATPase) enriched to the upper phase, and markers for ER (BiP) and tonoplast (VM23) enriched to the lower phase. Membrane vesicles containing ETR1 enriched in the lower phase, indicating that ETR1 is not localized to the PM. A good correlation between the distribution of ETR1 and the ER marker BiP was observed in several independent experiments, consistent with ER-localization.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Biochemical fractionation of Arabidopsis membranes showing co-fractionation of ETR1 with the ER. Arabidopsis membranes were fractionated then analyzed by immunoblot for ETR1, H+-ATPase (PM marker), BiP (ER marker), and VM23 (vacuole marker). A, structure of ETR1 showing location of transmembrane domains (black bars), GAF domain (diamond), histidine kinase domain (square), and receiver domain (oval). B, aqueous two-phase partitioning. Samples (5 µl) from the upper (U) and lower (L) phases were analyzed after partitioning. C, sucrose density-gradient centrifugation. Microsomal membranes were fractionated over 20-50% (w/w) Suc gradients run in the presence of Mg (+) to stabilize membrane-associated proteins or in absence of Mg () to dissociate membrane-associated proteins. Samples (20 µl) of each fraction were analyzed by immunoblot. Thylakoid membranes were identified spectrophotometrically. Golgi membrane fractions were identified based on Triton-stimulated UDPase activity.

To resolve the membrane location of ETR1, sucrose density-gradient centrifugation was performed with Arabidopsis microsomes. Centrifugation was performed in the presence and absence of Mg²⁺. Association of ribosomes with the ER is Mg²⁺-dependent, so removal of Mg²⁺ results in dissociation of ribosomes from the ER and a diagnostic redistribution of ER from higher to lower density on the gradient (21). Fractions from the sucrose gradient were analyzed by immunoblot for the presence of ETR1 as well as for markers specific for PM, tonoplast, and ER (Fig. 1C). ETR1 co-fractionated with the ER marker BiP and exhibited the same diagnostic Mg²⁺-dependent density shift from 29-36% to 38-44% (w/w) sucrose observed with the ER marker. A small amount of BiP, an ER resident chaperone, is also present at the top of the gradient and arises due to release of some soluble contents of the ER lumen. The distribution of ETR1 could be differentiated from the plasma membrane marker (H⁺-ATPase), the tonoplast marker (VM23), and the chloroplast thylakoid marker (chlorophyll absorbance), which did not demonstrate the same Mg²⁺-induced shift. ETR1 could be differentiated from the Golgi marker (latent UDPase), which had a broader distribution than ETR1 in the Mg²⁺-containing gradient.

Cytological Analysis of ETR1 Localization-- To cytologically corroborate ER localization for ETR1, we performed immunogold electron microscopy. For this purpose a polyclonal antibody against ETR1 was affinity-purified to monospecificity (Fig. 2). Due to low native levels of ETR1 in Arabidopsis, we used a transgenic line transformed with an additional genomic copy of the ETR1 gene (tETR1) for cytological studies (22). Transgenic expression of ETR1 resulted in a 4-fold increase in the level of immunodetectable ETR1 (Fig. 2). The increased expression of ETR1 does not affect receptor localization based on analysis by sucrose density gradient centrifugation (results not shown). In photomicrographs of mesophyll cells from Arabidopsis leaves, gold granules were found on the ER (Fig. 3, A-D). The ER was identified on the basis of associated ribosomes and by immunogold localization of ACA2 (Fig. 3, E-F), a transmembrane Ca²⁺ pump previously demonstrated to be ER-localized (16). Gold granules representing ETR1 were not detected in context of the PM except where the ER contacted the PM, nor were gold granules detected above background levels on other membrane systems such as mitochondria, chloroplasts, vacuoles, and the Golgi apparatus. Localization of ETR1 to the ER was confirmed by control experiments performed by omitting the anti-ETR1 antibody, by replacing it with preimmune antibody, and by probing the etr1-7 line (23) that lacks immunodetectable ETR1.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Monospecificity of anti-ETR1 antibody. Immunoblot analysis is shown for samples (20 µg) of soluble (s) and membrane (m) fractions isolated from wild-type Arabidopsis (WT), the etr1-7 loss-of-function mutant that lacks full-length ETR1, and from a transgenic line expressing an additional copy of the native ETR1 gene (tETR1). Migration positions of molecular mass markers are indicated in kilodaltons.

View larger version (150K): [in this window] [in a new window]   Fig. 3.   Localization of ETR1 to the ER by immunoelectron microscopy. Immunogold labeling of ETR1 (A-D) and ACA2 (E and F) in leaf mesophyll cells. C, D, and F are 2-fold enlargements of sections from A, B, and E, respectively. ETR1 labeling is confined to the rough ER as evidenced by the associated ribosomes. ETR1 labeling is absent from the PM, cell wall (cw), mitochondria (m), and vacuole (v). ACA2 labeling is present on the ER and is absent from the PM, cell wall (cw), chloroplast (c), and vacuole (v). Examples of gold granules (solid arrowheads) and ribosomes (open arrowhead) are indicated. A, B, C, D, E, and F contain 7, 5, 3, 3, 13, and 3 gold particles, respectively. Scale bar = 0.25 µm.

Efforts to visualize ETR1 by use of fusions to green fluorescent protein (GFP) were unsuccessful. Transient expression in Arabidopsis protoplasts of an ETR1-GFP fusion, driven by the cauliflower mosaic virus 35S promoter (35S::ETR1-GFP), could not be interpreted due to formation of large intracellular aggregates of fluorescence (results not shown). Stable expression of 35S::ETR1-GFP in Arabidopsis resulted in expression at close to native ETR1 levels, and the fluorescence was insufficient for visualization.

Stability of ETR1 Association with the ER-- To determine whether the ER localization reflects stable retention of ETR1 at the ER membrane, we treated Arabidopsis plants with the protein biosynthesis inhibitor cycloheximide. Use of cycloheximide has been demonstrated to be an effective means to clear transmembrane and secretory proteins in transit through the secretory pathway (24, 25). Preliminary experiments indicated that ETR1 was a long-lived protein, allowing use of this experimental approach. Immunoblot analysis of sucrose density-gradient fractions revealed that ETR1 was still ER-associated after a 6-h cycloheximide treatment based on the Mg²⁺-dependent density shift (Fig. 4), indicating that ETR1 was retained at the ER and did not migrate to the PM.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Effect of cycloheximide treatment upon localization of ETR1. Arabidopsis plants were treated with 300 µM cycloheximide for 6 h. Membranes were fractionated by sucrose density-gradient centrifugation in the presence (+) and absence () of Mg2+. Immunoblot analysis on 20 µl of each fraction was performed with antibodies against ETR1, the H+-ATPase (PM marker), and ACA2 (ER marker).

Localization of receptors may be affected by ligand binding (26). The endogenous levels of ethylene in plant tissue are not saturating because one does not observe the gross morphological changes associated with increased levels of ethylene (3). Therefore, to examine localization of ethylene receptors in an ethylene-bound state, plants were treated with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) for 24 h prior to membrane isolation. Treatment with 50 µM ACC is above the level needed to induce ethylene-mediated responses in Arabidopsis (27) and, based upon the dose dependence for ethylene binding by the ethylene receptor ETR1 (6), should produce sufficient ethylene to yield greater than 90% occupancy of receptor-binding sites. Immunoblot analysis of sucrose density-gradient fractions indicated that ETR1 isolated from ACC-treated plants was ER-associated based on the Mg²⁺-dependent density shift (Fig. 5A).

View larger version (63K): [in this window] [in a new window]   Fig. 5.   Effect of ligand binding upon localization of ETR1. Membranes were isolated from wild-type plants treated with 50 µM ACC for 24 h (A) or from the etr1-1 mutant line that contains a mutant receptor incapable of binding ethylene (B). Membranes were fractionated by sucrose density-gradient centrifugation in the presence (+) and absence () of Mg2+. Immunoblot analysis on 20 µl of each fraction was performed with antibodies against ETR1, the H+-ATPase (PM marker), and ACA2 (ER marker).

We also considered the formal possibility that a receptor lacking bound ligand might localize to a membrane system other than the ER. Inhibitors of ethylene biosynthesis exist, but these reduce rather than eliminate ethylene production in Arabidopsis (28). We therefore took a mutant-based approach to examine localization of an ethylene receptor incapable of binding ethylene. For this purpose, the ethylene-insensitive mutant etr1-1 was used. The etr1-1 mutation results in the change of a single amino acid (C65Y) within the second transmembrane domain of the receptor, disrupting binding of a copper co-factor necessary for liganding ethylene (6, 11). Immunoblot analysis of sucrose density-gradient fractions indicated that the etr1-1 mutant receptor was predominantly ER-associated based on the Mg²⁺-dependent density shift (Fig. 5B). A small amount of etr1-1 did not demonstrate a Mg²⁺-dependent density shift and may represent etr1-1 protein that has escaped the ER and now resides at the PM or at another membrane. Overall, our results indicate that the ligand-bound status of ETR1 has little effect upon the localization of ETR1 within the secretory system.

Determinants for Localization Are in the Amino-terminal Half of ETR1-- Several amino acid motifs have been implicated in the ER retention and retrieval of plant proteins. These include the cytosolic di-lysine motif, KKXX-COOH found in type-1 membrane proteins and the (H/K)DEL-COOH motif found in soluble proteins (29-31). ETR1 and other members of the ethylene receptor family in Arabidopsis lack these known ER localization motifs. To gain information on the localization requirements for ETR1, we examined the subcellular localization of ETR1-(1-349), a truncated version of the receptor that contains the amino-terminal 349 amino acids (full-length ETR1 = 738 amino acids) (20). ETR1-(1-349) was transgenically expressed in the etr1-7 genetic background of Arabidopsis that contains a loss-of-function mutation in the endogenous ETR1 gene. Analysis by sucrose density-gradient centrifugation supported ER localization of ETR1-(1-349) based on the Mg²⁺-dependent density shift (Fig. 6), indicating that determinants for ER targeting and retention are contained within the amino-terminal half of ETR1.

View larger version (44K): [in this window] [in a new window]   Fig. 6.   The truncated ethylene receptor ETR1-(1-349) localizes to the ER. Membranes were isolated from a line of etr1-7 that transgenically expresses the truncated receptor ETR1-(1-349). Sucrose gradient fractionation and immunoblot analysis on 20 µl of each fraction was performed. ETR1-(1-349) protein was detected using an antibody generated against amino acids 165-400 of ETR1 (10).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report here several independent lines of evidence demonstrating that the ethylene receptor ETR1 predominantly localizes to and is retained at the Arabidopsis ER. We cannot rule out that low levels of the receptor (e.g. 1%) may be present at other membrane locations, but if so, these are unlikely to be major contributors to ETR1-mediated ethylene perception for the following reasons. First, the pool of ETR1 is predominantly ER-localized based on all methods of analysis we employed. Second, we observed no change in ER localization following a cycloheximide chase; nor did we observe any substantial ligand-dependent change in localization. Third, due to the solubility of ethylene in aqueous and lipid environments (3), ethylene can readily access an intracellular pool of receptors (i.e. no transporter need be hypothesized for movement of ethylene across the PM to reach an ER-localized receptor). Thus, our data support the ER as the site of action for ethylene binding to the receptor ETR1. This localization is consistent with studies, performed before the identity of the ethylene receptors was known, in which ethylene-binding sites were reported at the ER and protein bodies of Phaseolus vulgaris L. (kidney bean) (32, 33).

ER localization presents several advantages for signal transduction. It is energetically efficient, as the receptor is not exported all the way through the secretory system to the plasma membrane. Receptors are rapidly delivered to their site of action, which may be important for members of the Arabidopsis ethylene receptor family such as ERS1, ERS2, and ETR2 whose expression is induced by ethylene (12). It also potentially allows for local regulation of ER-based processes such as Ca²⁺ release and protein secretion, Ca²⁺ having been previously implicated in plant ethylene responses (34-36). ETR1 could also potentially interact with the membrane-associated proteins RAN1 and EIN2 (37-39), both of which are implicated in ethylene signal transduction but whose subcellular localization is unknown. RAN1 is a copper transporter thought to deliver the copper co-factor required for ethylene binding by the receptors (37, 39). EIN2 is similar to the Nramp family of cation transporters, although no transport ability has been demonstrated for the protein (38).

Entrance into the secretory pathway and/or ER localization may be due in part to sequences present in the ancestral ethylene receptor. Ethylene receptors are thought to have originated with the endosymbiont cyanobacterium that gave rise to the chloroplast, because the cyanobacterium Synechocystis contains a protein slr1212 with similarity to the plant ethylene receptors (11). The slr1212 polypeptide contains three predicted transmembrane domains with 38% amino acid sequence identity to those of ETR1 and has the capacity to bind ethylene (11). Over evolutionary time, intracellular gene transfer from chloroplast to nucleus has occurred (40). Such transfers would typically be expected to result in "mis-localization" of the gene products, which lack transit sequences for retargeting back to the chloroplast. Transfer of a gene for the ancestral ethylene receptor from chloroplast to nucleus could therefore result in localization of the protein to a new membrane system, entrance into the secretory system representing one possibility as the mechanism for targeting to the ER is similar to that found in bacterial export systems (41).

Sequences required for both targeting to the secretory system and retention at the ER are contained within the amino-terminal half of ETR1. Other members of the ethylene receptor family, notably ETR2, ERS2, and EIN4, contain predicted signal sequences for entry into the secretory system (8), although whether these receptors are retained at the ER is currently unknown. ETR1 does not contain a predicted signal sequence and thus information for targeting to the secretory system is likely to be contained within its first transmembrane segment (42), which would in this case function as an uncleaved signal sequence. There is only limited information on sequence determinants for the retention of membrane proteins at the ER, the best known being the cytosolic di-lysine motif KKXX-COOH found in type-1 membrane proteins of plants, mammals, and yeast (31, 43, 44). This sequence is lacking in ETR1 and the other members of the Arabidopsis ethylene-receptor family, thereby supporting the existence of as yet unidentified sequences responsible for ER retention of the ethylene receptors.

The ethylene receptor ETR1 represents one of only a few ER-localized receptors that has been identified in eukaryotes. The transmembrane receptor Ire1 mediates the unfolded protein response in yeast, mammals, and plants, its ligands being unfolded proteins that accumulate in the ER lumen (45, 46). ABP1, a binding protein for the plant hormone auxin, is also predominantly ER-localized (47, 48). ABP1 is a soluble protein found in the ER lumen, localization being mediated in part by a KDEL sequence present at the carboxyl terminus. The function that ABP1 performs in the ER is still debated (49), but it has been hypothesized to act as a ligand-mediated chaperone (50). The finding that receptors for two of the best-characterized plant hormones are present at the ER is suggestive that the ER plays a central role in perception and signal transduction of plant hormones.

	ACKNOWLEDGEMENTS

We thank J. Collins, E. Hrabak, A. Klein, and the Schaller laboratory for critical reading of the manuscript, M. Sussman, J. Harper, M. Chrispeels, and M. Maeshima for antibodies, and E. Hrabak and J. Sheen for assistance on localization.

	FOOTNOTES

^* This work was supported by National Science Foundation Grant MCB-9982510 and is Scientific Contribution No. 2115 from the New Hampshire Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Dept. of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824. Tel.: 603-862-0565; Fax: 603-862-4013; E-mail: egs@cisunix.unh.edu.

Published, JBC Papers in Press, March 26, 2002, DOI 10.1074/jbc.M201286200

	ABBREVIATIONS

The abbreviations used are: PM, plasma membrane; ER, endoplasmic reticulum; GST, glutathione S-transferase; GFP, green fluorescent protein; ACC, 1-aminocyclopropane-1-carboxylic acid,.

	REFERENCES

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