Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis

Abstract

ETR1 represents a prototypical ethylene receptor. Homologues of ETR1 have been identified in Arabidopsis as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants. The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria. The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione S-transferase and affinity purified. Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP. The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine. Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain. Truncations were used to delineate the region required for histidine kinase activity. An examination of cation requirements indicated that ETR1 requires Mn2+ for autophosphorylation. These results demonstrate that higher plants contain proteins with histidine kinase activity. Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.

Figure 1

(A) Domains of the ETR1 protein. Hydrophobic, histidine kinase, and response-regulator domains are indicated. H indicates histidine-353, the putative phosphorylation site. G1 indicates position of the G1 box within the putative kinase domain. D indicates aspartate-659, a potential phosphorylation site. (B) Versions of ETR1 expressed as GST fusions in yeast. For truncations, the first and last amino acids of the expressed region are indicated, the full-length ETR1 protein being 738 amino acids long. For site-directed mutations, single letter abbreviations for amino acids Ala (A), Asn (N), Asp (D), Gln (Q), Gly (G), and His (H) are used.

Figure 2

(A) Purification of GST-ETR1(164–738) from yeast. SDS/PAGE profiles of proteins are shown from the soluble fraction, and after affinity purification of GST-ETR1(164–738) by binding to glutathione-agarose beads. Proteins are stained with Coomassie blue. Migration positions of molecular mass markers are indicated in kDa. (B) In vitro phosphorylation of GST-ETR1(164–738). Wild-type (WT) and mutant versions of the fusion protein were examined for the ability to autophosphorylate. The site-directed mutations were His-353–Gln (H), Asp-659–Asn (D), and in the G1 box (G1) of ETR1. Affinity- purified protein was incubated with ³²P-ATP, subjected to SDS/PAGE, then transferred to nylon membrane (in vitro). Proteins were sequentially treated with alkali (NaOH) and acid (HCl). After each treatment, incorporated phosphate was visualized by autoradiography. Finally, protein was visualized by Western blot using a polyclonal antibody against ETR1 (Ab).

Figure 3

In vitro phosphorylation of truncated versions of ETR1. GST fusions with either ETR1(164–609) or ETR1(333–609) were examined for the ability to autophosphorylate. Both wild-type (WT) and the His-353–Gln (H) site-directed mutation of ETR1 were tested in each case, and an autoradiograph of the alkali-resistant phosphorylation is shown (³²P). Relative size of the phosphorylated proteins, as based on molecular mass standards, is indicated in kDa. After autoradiography, the presence of each fusion protein was confirmed by Western blot using a polyclonal antibody directed against GST (Ab).

Figure 4

Cation dependence for autophosphorylation of ETR1. GST fusions with either ETR1(164–738) or ETR1(164–609) were examined for autophosphorylation in the presence of various cations (³²P). Wild-type (WT) and the His-353–Gln (H) site-directed mutation of ETR1 were tested with 5 mM MnCl₂, 5 mM MgCl₂, 5 mM CaCl₂, or a mixture of 5 mM MnCl₂ and 5 mM MgCl₂. After autoradiography, the presence of each fusion protein was confirmed by Western blot using a polyclonal antibody directed against GST (Ab).

Figure 5

pH dependence for autophosphorylation of ETR1. GST-ETR1(333–609) was incubated with ³²P-ATP at the indicated pH, subjected to SDS/PAGE, and transferred to nylon membrane. Alkali-resistant phosphate was visualized by autoradiography and quantified densitometrically. For pH 4.9 to 6.9 (•), the kinase assay mixture was buffered with 50 mM Mes, 100 mM Tris. For pH 6.9 to 8.3 (○), the kinase assay mixture was buffered with 100 mM Tris. The data were fit to a sigmoidal curve using sigmaplot. Data points represent the mean of duplicate samples.