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

Abstract

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...

Acclimation to soil flooding — sensing and signal-transduction

“…In plants, ethylene is perceived by a family of receptor molecules located in the endoplasmic reticulum (ER) (Chen et al, 2002). These receptor molecules share strong homology with bacterial two-component regulators (Chang et al, 1993), and are by default functionally active and are switched to an off-state by ethylene binding to the N-terminal transmembrane part of the dimerized molecule.…”

Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis

“…Immnunoblot assays were performed as described (Guo and Ecker 2003) with minor modifications. Membrane proteins were extracted according to Chen et al (2002); protein samples were mixed with 23 SDS-PAGE sample buffer, heated for 3 min at 90°C, and cooled on ice for 2 min. The proteins were fractionated by 4%-12% gradient Bis-Tris Novex precast gels (Invitrogen), transferred onto nitrocellulose filter and the blot was probed with anti-EIN2 or anti-H + -ATPase antibody (kindly provided by Dr M.J. Chrispeels).…”

“…Through these screens, many ethylene mutants have been obtained, including the ethylene-insensitive mutants etr1, ein2, ein3, ein5 (Bleecker et al 1988;Guzman and Ecker 1990;Roman et al 1995); the ethylene-overproducing mutants eto1, eto2, eto3, and the ethylene constitutive response mutant ctr1 (Guzman and Ecker 1990;Kieber et al 1993). Initial studies of these mutants have revealed a mostly linear framework for the ethylene-signaling pathway, leading from ethylene perception at the membrane to transcriptional activation in the nucleus (Stepanova and Ecker 2000;Chen et al 2002;Guo and Ecker 2004).…”

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

“…In Arabidopsis, ethylene is perceived by a family of five receptors that share homology to bacterial twocomponent His kinase receptors that are localized to the endoplasmic reticulum (ER) and Golgi membranes and form multimeric complexes thought to be composed of homoreceptor and heteroreceptor dimers (Chang et al, 1993;Hua et al, 1995Sakai et al, 1998;Chen et al, 2002Dong et al, 2008;Gao et al, 2008). Loss-of-function analyses indicate that the receptors act in a semiredundant manner to negatively regulate ethylene responses, although the ETR1 receptor appears to play more of a significant role in mediating ethylene responses than the other Arabidopsis receptors Cancel and Larsen, 2002;Hall and Bleecker, 2003;Wang et al, 2003;Binder et al, 2006;Qu et al, 2007).…”