Molecular association of the Arabidopsis ETR1 ethylene receptor and a regulator of ethylene signaling, RTE1.

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 µM). These findings indicate that a high affinity association of RTE1 and ETR1 is important in the regulation of ETR1.

Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy.

The plant ethylene receptor ETR1, which shows substantial sequence homology to typical bacterial histidine kinases, is involved in the coordination of several growth and development processes. Fluorescence polarization studies presented here demonstrate a specific interaction of ETR1 with the histidine-containing transfer protein AHP1, supporting the idea that a phosphorelay module is involved in ethylene signaling. The sensitive assay employed in our studies allows analysis of protein-protein interactions in a homogenous aqueous environment, exact control of external parameters, and quantitative analysis of the affinity and stability of the complex. Thereby it provides the basics for a more quantitative elucidation of phosphorelay modules acquired in phytohormone signaling.

Phosphorylation alters the interaction of the Arabidopsis phosphotransfer protein AHP1 with its sensor kinase ETR1.

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1-AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta.