Molecular Association of the Arabidopsis ETR1 Ethylene Receptor and a Regulator of Ethylene Signaling, RTE1

Abstract

The plant hormone ethylene plays important roles in growth and development. Ethylene is perceived by a family of membrane-bound receptors that actively repress ethylene responses. When the receptors bind ethylene, their signaling is shut off, activating responses. REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) encodes a novel membrane protein conserved in plants and metazoans. Genetic analyses in Arabidopsis thaliana suggest that RTE1 promotes the signaling state of the ethylene receptor ETR1 through the ETR1 N-terminal domain. RTE1 and ETR1 have been shown to co-localize to the endoplasmic reticulum (ER) and Golgi apparatus in Arabidopsis. Here, we demonstrate a physical association of RTE1 and ETR1 using in vivo and in vitro methods. Interaction of RTE1 and ETR1 was revealed in vivo by bimolecular fluorescence complementation (BiFC) in a tobacco cell transient assay and in stably transformed Arabidopsis. The association was also observed using a truncated version of ETR1 comprising the N terminus (amino acids 1-349). Interaction of RTE1 and ETR1 was confirmed by co-immunoprecipitation from Arabidopsis. The interaction occurs with high affinity (K d , 117 nM) based on tryptophan fluorescence spectroscopy using purified recombinant RTE1 and a tryptophan-less version of purified recombinant ETR1. An amino acid substitution (C161Y) in RTE1 that is known to confer an ETR1 loss-of-function phenotype correspondingly gives a nearly 12-fold increase in the dissociation constant (K d , 1.38 M). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.The gaseous plant hormone ethylene is important in regulating many aspects of growth and development, including fruit ripening, senescence, abscission, and stress responses (1). There are a number of known components in ethylene signaling that form a pathway starting with the perception of ethylene and leading to gene expression changes (2, 3). Arabidopsis thaliana has five homologous ethylene receptors with sequence similarity to histidine protein kinase receptors (4 -7). The receptors appear to be largely redundant in ethylene signaling, although the ETR1 ethylene receptor has a more predominant role (8 -10). The N terminus of the ethylene receptors comprises an ethylene-binding domain (11-13) consisting of three membrane-spanning domains localized at the endoplasmic reticulum (ER) (14 -16) and the Golgi apparatus (16). The cytosolic portion of the receptors exhibits histidine and/or serine/threonine protein kinase activity in vitro (17, 18) and control of autokinase activity by ethylene was demonstrated by in vitro phosphorylation studies using purified full-length ETR1 (19). However, the molecular mechanism of ethylene receptor signaling is still unknown, particularly as protein kinase activity appears to be largely dispensable for ethylene receptor signaling (8, 9).The ethylene receptors are negative regulators of ethylene response, repressing responses in the absence of ethylene (10,20) with the N-terminal domain contr...

Differential Control of Ethylene Responses byGREEN-RIPEandGREEN-RIPE LIKE1Provides Evidence for Distinct Ethylene Signaling Modules in Tomato

“…Genetic analysis indicates that AtRTE1 functions at or upstream of the ethylene receptors but acts independently of the RAN1 copper transporter (Resnick et al, 2006. In support of this hypothesis, AtRTE1 colocalizes with ETR1 in the ER and Golgi membranes and interacts with the N-terminal region of the ETR1 and ERS1 receptors (Zhou et al, 2007;Dong et al, 2008Dong et al, , 2010. Interestingly, mutations at the rte1 locus suppress ethylene insensitivity mediated by a subset of, but not all, etr1 mutant alleles, and reduced ethylene sensitivity mediated through AtRTE1 overexpression is primarily dependent on the presence of ETR1 and not the other ethylene receptors (Resnick et al, 2006Zhou et al, 2007).…”

“…The ethylene receptors are known to form large heteromeric complexes, and interaction experiments between the ethylene receptors and additional signaling components suggest that these complexes likely contain AtRTE1, EIN2, and CTR1 and occur within the ER and Golgi membranes (Clark et al, 1998;Chen et al, 2002Gao et al, 2003Gao et al, , 2008Bisson et al, 2009;Dong et al, 2010). Our data indicate that SlGR, SlGRL1, and SlGRL2 are localized primarily to the Golgi membranes at steady state, although at present we cannot completely exclude the possibility that these proteins are also localized to additional organelles ( Fig.…”

“…This study has identified variation in the control of ethylene responses by members of the GR/RTE1 gene family in tomato, revealing that individual members have evolved distinct abilities to influence ethylene responses and providing evidence for the existence of multiple ethylene signaling modules in tomato. Determination of the exact identity of these modules will require additional experimentation but, based on studies of Arabidopsis (Dong et al, 2010), may involve specific SlGR-ethylene receptor and SlGRL1-ethylene receptor interactions. The sequence variation between SlGR and SlGRL1 and their putative orthologs provides a tool to investigate this hypothesis and the role of these proteins in influencing specific ethylene responses.…”

“…This heterogeneity may lead to the formation of distinct signaling modules that produce diverse outputs. A model has been proposed in which AtRTE1 is required to maintain the ETR1 receptor in the "on signaling state" to inhibit ethylene responses, and biochemical evidence suggests that this occurs through a direct protein-protein interaction Dong et al, 2010).…”

EIN2 mediates direct regulation of histone acetylation in the ethylene response

“…Each ethylene receptor has an N-terminal transmembrane domain, and the receptors form dimers that bind ethylene via a copper cofactor RAN1 (10,11). Signaling from one of the receptors, ETR1, induces its physical association with the ER-localized protein RTE1 (12). The ethylene receptors function redundantly to negatively regulate ethylene responses (5) via a downstream Raf-like protein kinase called CTR1 (13,14).…”